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==== Front Cancer Cell IntCancer Cell International1475-2867BioMed Central London 1475-2867-4-51531571110.1186/1475-2867-4-5Primary ResearchPotential mechanism of phytochemical-induced apoptosis in human prostate adenocarcinoma cells: Therapeutic synergy in genistein and β-lapachone combination treatment Kumi-Diaka James [email protected] Simone [email protected] Alex [email protected] Jayann [email protected] Department of Biological Sciences, Schmidt College of Science, Florida Atlantic University @ Davie, 2912, College Avenue, Davie FL. 33314, USA2 Rambaugh-Goodwin Cancer Research Institute, Sunrise, FL. USA2004 17 8 2004 4 5 5 16 2 2004 17 8 2004 Copyright © 2004 Kumi-Diaka et al; licensee BioMed Central Ltd.2004Kumi-Diaka et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Prostate cancer is the second leading cause of male death in the United States. The incidence increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of the cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited; and hence treatment has only been partially successful. The objective of the current studies was to examine the role of caspase 3 (CPP32) and NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of genistein-and β-lapachone (bLap)-induced apoptosis in human prostate carcinoma cells PC3. Results Both genistein and bLap produced dose-dependent growth inhibition and treatment-induced apoptosis in PC3. Treatment with caspase 3 inhibitor, DEVD-fmk before exposure to genistein, significantly inhibited caspase 3 expression and treatment-induced apoptosis; implicating CPP32 as the main target in genistein-induced apoptosis in PC3. Contrary to this observation, inhibition of CPP32 did not significantly influence bLap-induced apoptosis; implying that the major target of bLap-induced apoptosis may not be the caspase. Treatment with NQO1 inhibitor, dicoumarol (50 μM), prior to exposure of PC3 to bLap led to significant decrease in bLap toxicity concurrent with significant decrease in treatment-induced apoptosis; thus implicating NQO1 as the major target in β-lapachone-induced apoptosis in PC3. In addition, the data demonstrated that NQO1 is the major target in bLap-genistein (combination)-induced apoptosis. On the contrary, blocking NQO1 activity did not significantly affect genistein-induced apoptosis; implying that NQO1 pathway may not be the main target for genistein-induced apoptosis in PC3 cells. Furthermore, blocking NQO1 and CPP32 did not confer 100% protection against genistein-induced or bLap-induced apoptosis. Conclusion The data thus demonstrate that both genistein-and bLap-induced apoptosis are mostly but not completely dependent on CPP32 and NQO1 respectively. Other minor alternate death pathways may be involved. This suggests that some death receptor signals do not utilize the caspase CPP32 and/or the NQO1 death pathways in PC3. The demonstrated synergism between genistein and bLap justifies consideration of these phytochemicals in chemotherapeutic strategic planning. ==== Body Background Prostate cancer is the most common non-skin malignancy and the second leading cause of male death in the United States [1]. The incidence of prostate cancer increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of prostate cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited. The problem often faced with the clinical management of prostate cancer is derived not only from the fact that no single gene or molecule can serve as a reliable marker [2,3], but also that there is still no effective therapeutic regimen available without serious, sometimes fatal side effects. Unfortunately, at the time of clinical diagnosis, human prostate cancers mostly present themselves as heterogeneous entities – hormone-dependent and hormone-independent, and proliferating and non-proliferating. The tumor re-growth that occurs after post-treatment remission is largely due to progression of initially androgen-dependent to androgen-independent cancer cell [4] and/or non-proliferating to proliferating tumor cells. Therefore chemotherapeutic strategies should focus on eradicating all cancer cells irrespective of state of growth or sensitivity to hormone. This calls for a search for drug combination treatment that works through different mechanism of action. The facts that prostate cancer cells retain the inherent apoptotic machinery potentially subject them to an appropriate efficacious chemotherapeutic intervention. The molecular mechanism(s) and intracellular mediators of both spontaneous-and treatment-induced apoptosis are not fully elucidated. However, evidence from several research investigations seem to indicate that a variety of stimuli, including physiological, pathologic, environmental or cytotoxic, can trigger the process of apoptosis in many mammalian cells [5,6], and that both apoptosis and necrosis may share some upstream events in the molecular pathways that lead to induction of apoptosis [7-11]. An emerging strategy for cancer chemotherapy is the choice of drugs that induce apoptosis and/or disruption of angiogenesis with eventual elimination of the cancer. It is suggested that blocking of caspase activation in an apoptotic process may divert apoptotic cell death to a necrotic demise [10]; implying that apoptosis and necrosis may share some upstream events in the molecular pathways of apoptosis induction. Among the dietary phytochemicals of potential therapeutic significance, are genistein isoflavone and β-lapachone, both of which induce apoptosis and also inhibit angiogenesis (genistein) in an array of cancer cells [6,12,13]. Genistein isoflavone [4',5',]-trihydroxyisoflavone) is a metabolite of soy [14] and has a heterocyclic, diphenolic structure similar to estrogen [14]. The phytochemical isoflavonoid family to which genistein belongs is a group of plant chemicals that resemble steroid estrogens and mimic their biological reactions [15,6,16]. Several clinical studies indicate that genistein has some chemoprotective and chemotherapeutic potential against many tumors, including prostate, breast, and colon cancers through several mechanisms of action including: apoptosis induction; modulation of cell cycle activity by arresting cell cycle at the G2-M stage [17]; inhibition of DNA topoisomerase-II and tyrosine protein kinase [18]; competitive inhibitor of ATP binding to the catalytic domain of tyrosine kinase [14,18]; stimulating the production of sex hormone-binding globulin (SHBG), which may lower the risk of hormone related cancers by decreasing the amount of free and active hormones in the blood [19,20]. The other phytochemical of potential therapeutic significance is β-lapachone [3, 4-dihydro-2, 2-dimethyl-2H-naphtol (1,2-b) pyran-5,6-dione], a simple plant product with a chemical structure different from currently used anti-cancer drugs. It has been previously demonstrated that the primary mode of cytotoxicity of β-Lapachone is through the induction of apoptosis [21,22]. Structural similarities between β-lapachone and other members of the naphthoquinone family, such as menadione, suggest that the enzyme, NAD(P)H:Quinone oxidoreductase enzyme (NQO1) may be involved in the activation or detoxification of β-lapachone [23-25]. While a number of in vitro effects of β-lapachone and genistein have been described, knowledge of the key intracellular targets of β-lapachone and genistein is limited. Recent reports have suggested that by β-lapachone-induced apoptosis is non-caspase mediated in breast [26] and prostate cancer cells [21,34], and that the cytotocxicity of this compound is dependent on the activity of NAD(P)H:Quinone oxidoreductase enzyme (NQO1/xip3) [26,27]. B-Lapachone has been shown to be an inhibitor of DNA repair that sensitizes cells to DNA-damaging agents [28,29]. It directly inhibits DNA topoisomerase I and II [30-32] and induces a cell-cycle delay in G1 and/or S phase followed by apoptotic and/or necrotic cell death [13]. The apoptosis induced by β-lapachone is p53 independent [21], and has been associated with upregulation of Bak as well as cleavage of caspase-7 [33] and caspase 3 in a variety of mammalian cells [13,33]. The objective of this study was to determine the potential chemosensitivity of human prostate adenocarcinoma, PC3 cells to β-lapachone and Genistein and the role of caspase 3 (CPP32) and NAD(P)H:Quinone oxidoreductase enzyme (NQO1) in the signaling of β-Lapachone and genistein-induced apoptosis in PC3 cells. The hypothesis is that combination treatment with the two phytochemicals will be strongly preventive and/or interceptive against prostate cancer by modulating epigenetic events (apoptosis) associated with the progression of active and latent cancer cells to clinical malignancy. Results Genistein and β-lapachone inhibit growth and proliferation of human prostate carcinoma cells, PC3 Human prostate carcinoma cells PC3, was used to determine the chemosensitivity of prostate cancer to genistein isoflavone and β-lapachone in vitro using Trypan blue exclusion, LDH and MTS bioassays. In single and combination treatments, both genistein and β-lapachone inhibited cell growth and decreased cell survival through induction of cell death [Figures. 1,2,3]. The data indicated that PC3 sensitivity to both single and combination treatment is dose-dependent, and that PC3 was significantly more sensitive (P < 0.05) to the combination treatment than to the single treatment; indicating a potential synergism between genistein and β-lapachone [Figures 2,3]. Figure 1 β-lapachone-induced growth inhibition in PC3. PC3 cells (1 × 104 cells/well) were cultured in 24-well plates for 48 hr to allow 85–90% confluence; treated with varyingconcentrations of bLap and assessed for post-treatment viabilitywith the MTS assay. Note the dose-dependent growth inhibitionin PC3. Data points represent means ± SEM of three independentexperiments performed in triplicates Figure 2 Genistein (Gn)/β-Lapachone combination treatment of PC3. Cells were treated as described in the methods and subjected to post-treatment viability with MTS colorimetric assay. Data points represent the means ± SEM of three independent experiments performed in triplicates. Figure 3 Single and combination of PC3 cells with genistein (Gn) and β-lapachone (bLap) βLap. Briefly, PC3 cells were seeded at 1 × 104 cells/well in 48-well MTP and co-cultured with Gn0-70 with/without bLap (1.2 μM); followed by determination of treatment-induced cytotoxicity as described in the methods. Data points represent means ± SEM of three experiments performed in triplicates Genistein and β-lapachone induce apoptosis in human prostate cancer cells Extensive cell death was observed in proliferating human prostate cancer cells after treatment with β-lapachone and genistein isoflavone. To determine if the treatment-induced cell occurred through cytotoxic necrosis and/or apoptosis, cells were harvested and assayed for apoptosis induction with Annexin V-FITC and TUNEL apoptosis assays to detect early and late apoptosis respectively. Aliquots of cells were also stained with acridine orange/ethidium bromide nuclear stain to distinguish between apoptotic and necrotic cells. The results revealed that in both single and combination treatments, cell death was mostly through apoptosis in a dose-dependent manner [Figures 4,5]. With increasing concentration of the agents, cell death through necrosis increased correspondingly. Furthermore, combination treatment induced significantly more apoptosis in PC3 (p <0.01) than individual treatment with either agent. Figure 4 βLap)-induced cell death in PC3 cells. PC3 cells were co-cultured with varying concentrations of bLap; and and the degree of treatment-induced apoptosis and/or necrosis assessed with the Annexin V-FITC assay, as described in the methods. Data points are means ± SEM of three independent experiments performed in triplicates. Figure 5 Combination treatment-induced cell death in PC3 cells. PC3 cells were co-cultured with varying concentrations of Gn (Gn0-70) with or without bLap (1.2 μM), and apoptotic/necrotic cell death assessed with theAnnexin V-FITC assay as described in the methods. Data points are means ± SEM of three independent experiments performed in triplicates Dicoumarol enhanced the survival of human prostate cancer cells (PC3) following single treatments with β-lapachone (bLap) and Gn/bLap combination but not in PC3 cells treated with genistein alone To determine the potential role of the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in PC3, the cells were exposed to Gn and bLap in the presence or absence of dicoumarol in single and combination treatments; and then assayed for apoptosis by the Annexin V-FITC and TUNEL assays. Dicoumarol is a specific inhibitor of NQO1. The results revealed that blocking NQO1 activity with dicoumarol (50 μM) significantly reduced bLap-induced apoptosis [Figure 6]; indicating that bLap-induced apoptosis requires involvement of NQO1 target. However, dicoumarol did not appear to have significant effect on Gn-induced apoptosis [Fig 7]; indicating that NQO1 did not play significant role in Gn-induced apoptosis. [Figures 6,7]. The degree of apoptosis induction was highest in the Gn-bLap combination treatment without inhibiting NQO1 activity with dicoumarol [Figure 7]; implying that a possible synergy between Gn and bLap may be due to NQO1 activity. Figure 6 Role of NQO 1 in β-Lapaphone-mediated apoptosis in PC3 cells. PC3 cells were treated with bLap alone or in combination with 50 μM dicoumarol (NQO 1 inhibitor) as described in the methods; and TUNEL assays performed to monitor apoptosis. Data points represent means + SEM of three independent experiments performed in triplicates. Figure 7 NQO1 is the main target in bLap/Gn-induced apoptosis in PC3 cells. Cells were treated with genistein (Gn) and Gn/bLap combination with or without 50 μM dicoumarol as described in the methods; and TUNEL assays performed to monitor apoptosis. Data points represent means ± SEM of three independent experiments performed in triplicates. Activation of CPP32 in genistein-induced apoptosis in PC3 but not in β-lapachone-induced apoptosis in PC3 To determine if apoptosis induced by β-lapachone and/or genistein involved activation of caspase 3 protease (CPP32), the PC3 cells, were subjected to treatments with Gn and/or bLap co-administered with or without CPP32 inhibitor (DEVD-fmk), and then cultured as previously described. Post-treatment apoptosis was determined as previously described. As shown in Figures 8 and 9, blocking the release of caspase 3 significantly decreased genistein induced apoptosis but not bLap-induced apoptosis; indicating the significant role of CPP32 in the molecular pathway of Gn-induced apoptosis; and minor involvement of CPP32 in bLap-induced apoptosis in PC3. Furthermore, blocking CPP32 activity did not significantly affect combination treatment-induced apoptosis (Figure not shown). Figure 8 Caspase-3 (CPP32) activity in genistein-induced apoptosis in PC3 cells. PC3 cells (2.5 × 103 cells/well) were cultured; then treated with/without 100 μM caspase inhibitor (zVAD-fmk) for 2 hr; and then with 10–70 μg/mL genistein for 4 hr as described in the methods. Cells were thenanalyzed for caspase (CPP32) activity and corresponding apoptosis in the cells. Data points were the means ± SEM of two independent experiments performed in triplicates. Figure 9 CPP32 is the major pathway in genistein-induced apoptosis in PC3 cells. PC3 cells (2.5 × 103 cells/well) were cultured in 48-well culture plates; treated with/without 100 μM caspase inhibitor (zVAD-fmk) for 2 hr; then with 1–8 μM β-Lapachone (bLap) for 4 hr as described in the methods. Cells were then analyzed for caspase (CPP32) activity and corresponding apoptosis. Data pointsare the means ± SEM of two independent experiments performed in triplicates Discussion and Conclusions In this study, we determined the role of caspase 3 (CPP32) and the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in human prostate adenocarcinoma, PC3 cells. Data from this study demonstrate significant inhibition of cell growth and proliferation in PC3 cells, with significant difference in chemosensitivity of PC3 to genistein and β-lapachone (P < 0.01). Furthermore, growth inhibition of PC3 cells strongly correlated with the MTS and LDH assay results. The pattern of response and percent post-treatment live cells was consistent with previous results [6,37-39]. The genistein-and bLap-induced morphological changes observed in the cells were identical in pattern but differed in severity at a given exposure time; indicative of differences in chemosensitivity of PC3 to genistein and β-lapachone. These observations were consistent with previous results [5,6]. Furthermore, previous studies have shown that β-lap [22] induces morphologic changes indicative of apoptosis in human breast cancer cells. Similar alterations in morphology including cell shrinkage and chromatin condensation in the PC3 cells following single and combination treatment with β-lapachone and genistein isoflavone. The present data also implicates caspase-3 protease, CPP32, in the molecular pathway of genistein-induced apoptosis in prostate PC3 cancer cells, consistent with previous investigations [10,11,39]. Using the caspase inhibitor DEVD-fmk, caspase activity was arrested concurrent with significant decrease in genistein-induced apoptosis in PC3 cells. However, it is noteworthy that inhibition of caspase did not confer 100% protection against genistein-induced apoptosis; implying alternative death pathways, which suggests that some death receptor signals do not utilize the caspase CPP32 death pathways in PC3. We have previously demonstrated the significant role of caspase-3 protease in the genistein-induced apoptosis pathway in both testes and prostate (PC3) cancer cells [38,39]. The present data indicate a possible alternate CPP32 pathway in bLap-induced apoptosis in PC3. However, unlike the observation in genistein-induced apoptosis, blocking the CPP32 activity with the specific caspase inhibitor, DEVD-fmk, did not significantly change the percentage of bLap-induced apoptosis in PC3 cells; indicating that CPP32 many not be the main death pathway of bLap-induced apoptosis in PC3 cells. Activation of the caspase 3 in bLap-induced apoptosis has been reported in previous studies [10]. The potential role of NAD(P)H:quinone oxydoreductase (NQO1) activity in genistein-and bLap-induced apoptosis in PC3 was investigated. Co-culture of PC3 cells with dicoumarol, a specific inhibitor of NQO1 activity, significantly reduced the cytotoxicity of β-Lapachone in PC3 cells, as reflected in the significant reduction in the percentage of treatment-induced apoptosis. Dicoumarol increased cell survival. These results implicate NQO1 as the main target in bLap-induced apoptosis in PC3, consistent with previous observations [26,34,40]. However, the fact that blocking of NQO1 did not confer 100% protection against induction of apoptosis indicates a possible alternate pathway in bLap-induced apoptosis. The present data indicate some involvement of caspase protease CPP32, though not with the same significance as NQO1. The activation of cysteine protease has been observed after bLap treatment [26]. Pink et al [26] reported activation of the cysteine protease in MCF-7 and T4D breast cancer cells in bLap-induced apoptosis. Contrary to the observation in bLap-induced apoptosis, blocking NQO1 activity did not significantly influence genistein-induced apoptosis in PC3 cells; implying NQO1 may not be the major target in genistein-induced apoptosis in PC3 cells. However, the overall data indicate a synergistic effect of genistein-bLap combination treatment of PC3 and, that the major target in the combination treatment-induced apoptosis in PC3 cells is NQO1. Investigation into genistein/bLap synergism in a number human cancer cells is on-going. Conclusion It is concluded from the data obtained that: i) both genistein and bLap exert growth inhibition effects in PC3 cells, with significant differences in chemosensitivity of PC3 to the two agents; ii) there is a manifestation of synergism between genistein-bLap combination treatment; iii) both genistein and bLap induce apoptosis in PC3 cells; iv) the major target in genistein-induced apoptosis in PC3 cells seems to be CPP32; v) the major molecular target in bLap-induced apoptosis in PC3 cells appear to be NQO1; vi) the major target in the genistein-bLap combination treatment-induced apoptosis appears to be NQO1; and vii) combination treatment appears significantly more efficacious than single treatments. More extensive studies are ongoing to delineate and clarify the molecular mechanisms underlying the combination effects. Materials and Methods Chemicals Genistein isoflavone (Indoline Chemical Co. Summerville, NJ, USA) was constituted in DMSO (dimethylsulfoxide) solvent as 10, 20, 30, 50 and 70 μg/ml solutions (G10-70) and frozen at -37°C until used. β-lapachone (Sigma Scientific (St. Louis, MO, USA) was constituted in DMSO solvent as 1, 2, 3, 5 and 8 μM/ml solutions (bL1-8) and stored at -37°C. Dicoumarol (Sigma Scientific St. Louis, MO, USA) was constituted in DMSO as 50 μM/ml and frozen at -37°C until used. The caspase 3 inhibitor, DEVD-fmk and substrate DEVD-afc were purchased from Biovision (Palo Alto, CA). Culture media (RPMI 1640), antibiotics, trypsin-EDTA, and other reagents were purchased from Sigma scientific (St. Louis, MO, USA). Cell lines Human prostate adenocarcinoma (PC3) was a generous donation from Rambaugh-Goodwin Cancer Research Institute, Plantation FL. Cell Culture To assess the chemosensitivity of human prostate cancer cells to single and combination treatment with genistein (Gn) and β-lapachone (bLap), cells were sub-cultured under 5% CO2 at 37°C for 48 hrs to reach 80–90% confluence. All cells were grown and maintained as monolayers in 25 m2 tissue culture flasks (Sigma Scientific, St. louis, MO, USA) in RPMI 1640 containing 15 mM HEPES, and supplemented with 0.45% glucose (w/w), 5.0% FBS and 100 U. mL-1 penicillin + 100 mg. ml-1 streptomycin. The cells were then harvested by gentle scraping with a cell scraper. The cell suspensions were then grown at a density of 2.5 × 103 cells/well in 24-well microtiter plates (MTP) for 36 hr to allow adherence. The supernatants were discarded and the agents (Gn or bLap) were added over a range of 5 cytotoxic concentrations in single and combination treatments. In preliminary studies with bL1-8, the IC50 ranged between 1.8–3 μM for a number of cells. Therefore in the present studies, 2.0 μM (bL2) was used in the combination studies with varying concentrations of genistein. All treatments were in triplicates. Dicoumarol was added to the cells and incubated for 4 hr before treatment with either genistein or β-lapachone alone or in combination. All plates were then incubated at 37°C in a humidified atmosphere of 5% CO2 in air for a maximum of 72 hr. At 12, 24 and 36 hr of incubation, 100 μl of the supernatant from each well was gently aspirated and replenished with 100 μl of fresh media. The supernatants were stored at -37°C until assayed for lactate dehydrogenase (LDH) enzyme activity. At 36 hr incubation, the cells were harvested by trypsinization with trypsin-EDTA, and processed for post-treatment metabolic activity using cell viability and apoptotic assays as described. A. Cell Viability Assays A1.1 MTS assay MTS assay depends on mitochondrial enzyme reduction of MTS solution to detect and determine cell viability. The MTS cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation. It is composed of solutions of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). MTS is bioreduced by the cells into formazan product that is soluble in cell culture medium. Following cell culture as described above, 100 μL of cells were harvested from each treatment group and added to a 96 MTP followed by addition of 20 μl of MTS (2.5 mg/mL: Sigma Chemical Co) stock solution to each well. After 2 hr incubation under standard conditions of 5% CO2 and 37°C, the purple formazan product (indicative of reduction of MTS) was visible. The absorbance was read on Multiskan biochromatic automated microplate reader (Multiskan, DC) at 490 nm. The signal generated (color intensity) is directly proportional to the number of viable (metabolically active) cells in the wells. Relative cell numbers can therefore be determined based on the optical absorbance (optical density, OD) of the sample. The blank values were subtracted from each well of the treated cells and controls; and the mean and standard error for each treatment (singles and combination) were calculated relative to the control: where AC = absorbance of the control (mean value): AT = absorbance of the treated cells (mean value) AB = absorbance of the blank (mean value) A1.2 Trypan Blue exclusion assay For the Trypan blue exclusion test, cells were treated and cultured as described. They were harvested and Trypan blue dye solution was added to the cell suspensions. Total cell counts and viable cell number (survival rate) were determined by a standard hemocytometer procedure. Live-viable cells were seen as colorless (impermeable to the dye due to intact cell membrane) and dead cells were seen as blue (permeable to dye due to disruption of cell membrane): A2. LDH assay Lactase dehydrogenase activity was measured by a non-radioactive protocol using the LDH cytotox kit (Cat. #. 1644 793: Boehringer-Mannheim GmbG, Bochemica). The previously frozen supernatants were thawed for LDH determination. Briefly, 100 μL/well of each cell-free supernatant was transferred in triplicate into wells in a 96-well microtiter plate (MTP) and 100 μL of LDH-assay reaction mixture (Kit: dye-catalyst mixture) added to each well. After 90 min incubation under standard conditions the absorbance of the color generated was read on Multiskan biochromatic automatic microplate reader at 490 nm. The mean absorbance/optical density (OD) for each treatment group was calculated. The blank values were subtracted from each well and the mean percent treatment-induced cytotoxicity for each cell line and treatment type (single and combination) was calculated as: Where: ABSexpt = mean absorbance from the treated cells: ABSlow = mean absorbance from controls (untreated cells)(spontaneous release of LDH) ABShi = mean absorbance from Triton X-100 treated cells (standard/maximum LDH release)(positive control). B. Detection of Treatment-induced Apoptosis Treatment-induced apoptosis was assessed by two independent assays, Annexin V-FITC assay and the DNA Fragmentation (TUNEL) assay. PC3 cells were treated and co-cultured with the test agents as previously described in this study; and the subjected to the apoptosis determination assays as below: B1.1 Annexin V-FITC assay Apoptosis-associated translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane in GC27 and K833 cells was assessed with the use of FITC-labeled Annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine; using AnnexinV-FITC Staining Kit (Boehringer Mannheim). Briefly, 100 μl aliquots of the previously prepared cell suspensions were centrifuged, and the cell pellets re-suspended in Annexin binding buffer, incubated with AnnexinV-FITC substrate; then cells were smeared onto microscope slides and either evaluated immediately with fluorescence microscope, or smears were fixed with 4% depolymerized paraformaldehyde and stored at -40°C for later examination as previously described [35]. Percentage of apoptosis in the cells was quantified based on morphological and fluorescence characteristics of apoptotic cells as previously described [5,35,36]. All tests were run in triplicates. B1.2 DNA Fragmentation (TUNEL) assay The presence of apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), using the ApopTagR kit (Boehringer Mannheim Co, Indianapolis, IN) as previously described [37]. The kit reagents detect apoptotic cells in situ by specific end labeling and detection of DNA fragments produced by the apoptotic process. To perform the TUNEL assay, slides of the PBS suspended cells were fixed with 4% paraformaldehyde for 30 minutes. The cells (slides) were then permeabilized with Triton X-100 at 4°C for 2 min; then flooded with TdT enzyme and digoxigenin-dUTP reaction buffer (TUNEL) reagent for 60 min in a humidity chamber at 37°C, washed with distilled water, incubated for 10 minutes with streptavidin-horseradish peroxidase complex. The stained mounted cells were examined at 100×, 200× and 400× magnification of the microscope (Olympus BH-2). Cell death was quantified by counting 150 cells in 5–7 separate fields of view per slide, and noting the percentage of apoptotic cells based on morphological appearance, as previously described [5,36]. C Potential mechanism(s) of action The potential involvement of caspase-3 protease (CPP32) and/or the enzyme NQO1 [NAD(P)H:quinone oxidoreductase] in the molecular pathways of β-lapachone-and/or genistein-induced growth inhibition and apoptosis in PC3 cells were determined, after treatment of the cells as already described. C1.1 Caspase-3 expression/activity in treatment-induced apoptosis In order to determine the potential role of caspase-3 proteases (CPP32) in the common pathways of β-lapachone and genistein-induced growth inhibition and apoptosis, human prostate cancer cell lines were treated as previously described above. The activity of caspase 3 was determined using a the fluorometric substrate DEVD-afc and caspase 3 inhibitor DEVD-fmk according to the protocol of the Caspase Activity Assay kit. Briefly, PC3 cells were treated and incubated as previously described. At 24, 48 and 72 hr cells were scrapped into suspension and centrifuged at 10,000 rpm for 10 min. The pellet was resuspended in 100 μl of lysis buffer and incubated at 4°C for 10 min, followed by centrifugation at 10,000 rpm for 10 min. Fifty μl aliquots of the supernatants were removed and placed in a 96-well microtiter plate (MTP) containing reaction buffer. The DEVD-afc substrate was added and the MTP was incubated at 37°C for 30 min. Activity was monitored with the linear cleavage and release of the afc side chain; and compared with a linear standard curve generated by the controls on the same MTP. C1.2 NQO1 Activity in treatment-induced apoptosis In order to determine the potential role of enzyme NQO1 [NAD(P)H:quinone oxidoreductase] in the molecular pathways of β-lapachone-and genistein-induced growth inhibition and apoptosis in human prostate cancer, PC3 cell lines were treated as previously described. Dicoumarol (3-3'-methylene-bis (4-hydroxycou-marin) is a commonly used inhibitor of NQO1, which competes with NADH or NADPH for binding to the oxidized form of NQO1. Dicoumarol thereby prevents reduction and activation of various target quinines like β-lapachone. The cells, cultured as previously described, were treated concomitantly in single and combination treatments of varying concentrations β-lapachone (bLap), genistein (gen), and bLap-Gn combination with and without 50 μM dicoumarol as previously described. The treated cells were harvested and tested for treatment-induced apoptosis by the methods previously described in this study. Authors' contributions JKD contributed 50% in all aspects of the research. All other authors contributed equally-50% All authors read and approved the final manuscript. Acknowledgments The authors acknowledge the generous offer of cell lines by the Rambaugh-Goodwin Cancer Research Institute at Plantation FL. and thank Florida Atlantic University for partial support of this research. ==== Refs Lynn AG Ries MS Stat Bite: Top 5 cancers for females and males in the U.S JNCI 1995 87 867 Zhau HE Pisters LL Hall MC Zhao LS Troncoso P Pollack A Chung LWK Biomarkers associated with prostate cancer progression J Cell Biochem Suppl 1994 19 208 216 7529853 Webber MM Waghray A Bello D Prostate-specific antigen, a serine protease, facilitates human prostate cancer cell invasion Clin Cancer Res 1995 1 1089 1094 9815898 Umekita Y Hiipakka RA Kokontis JM Liao S Human prostate tumor growth in athymic mice: Inhibition by androgens and stimulation by finasteride Proc Natl Acad Sci USA 1996 93 11802 11807 8876218 10.1073/pnas.93.21.11802 Kumi-Diaka J Nguyen V Butler A Cytotoxic potential of the phytochemical genistein isoflavone (4',5',7-trihydroxyisoflavone) and certain environmental compounds on testicular cell lines Biol Cell 1999 91 515 523 10572627 10.1016/S0248-4900(00)88208-8 Kumi-Diaka J Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and β-lapachone Biol Cell 2002 94 37 44 12000145 10.1016/S0248-4900(01)01175-3 Liu X Kim CN Yang J Jemmerson R Wang X Induction of apoptotic program in cell-free extracts requirement for dATP and cytochrome c Cell 1996 86 147 157 8689682 10.1016/S0092-8674(00)80085-9 Green Dr Reed JC Mitochondria and apoptosis Science 1998 281 1309 1312 9721092 10.1126/science.281.5381.1309 Janicke RU Ng P Sprengart ML Porter AG Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis J Biol Chem 1998 273 15540 15545 9624143 10.1074/jbc.273.25.15540 You-Zhi Li Chiang Li J Pinto AV Pardee AB Release of mitochondrial cytochrome C in both apoptosis and necrosis induced by b-Lapachone in human carcinoma cells Molecular Medicine 1999 5 232 239 10448645 Nicholson DW Thornburry NA Life and Death Decision Science 2003 299 214 215 12522239 10.1126/science.1081274 Li CJ Wang C Pardee AB Induction of apoptosis by β-lapachone in human prostate cancer cells Cancer Res 1995 55 3712 3715 7641181 Li CJ Li YZ Pinto AV Pardee AB Potent inhibition of tumor survival in vivo by β-Lapachone plus Taxol: combining drugs imposes different artificial checkpoints Proc Natl Acad Sci USA 1999 96 133 69 9874784 10.1073/pnas.96.1.133 Adlercreutz CH Goldin BR Gorbach SL Hockerstedt KA Watanabe S Hamalainen EK Markkanen MH Makela TH Wahala KT Soybean phytoestrogen intake and cancer risks J Nutr 1995 125 757S 770S 7884562 Kim H Peterson TG Barnes S Mechanism of action of the soy isoflavone, genistein: Emerging role for its effects via transforming growth factor signaling pathways Am J Clin Nutr 1998 68 1418S 1425S 9848510 Dixon RA Daneel Ferreira Genistein Phytochemistry 2002 60 205 211 12031439 10.1016/S0031-9422(02)00116-4 Matsukawa Y Marui N Sakai T Satomi Y Yoshida M Matsumoto K Nishino H Aoike A Genistein arrests cell cycle progression at G2-M Cancer Res 1993 53 1328 1331 8443813 Akiyama T Ishida J Nakagawa S Ogawara H Watanabe S Itoh N Shibuya M Fukami Y Genistein, a specific inhibitor of tyrosine-specific protein kinases J Biol Chem 1987 262 5592 5595 3106339 Messina MJ Persky V Setchell KDR Barnes S Soy intake and cancer risk: A review of the in vitro and in vivo data Nutr Cancer 1994 21 113 131 8058523 Mousavi Y Adlercreutz H Genistein is an effective stimulator of sex hormone binding globulin production in hepatocellular human liver cancer cells and suppresses proliferation of these cells in culture Steroids 1993 58 301 304 8212077 10.1016/0039-128X(93)90088-5 Planchon SM Wuerzberger S Frydman B Witiak DT Hutson P Church DR Wilding G Boothman DA β-lapachone-mediated apoptosis in human promyelocytic leukemia (HL-60) and human prostate cancer cells: a p53-independent response Cancer Res 1995 55 3706 3711 7641180 Wuerzberger SM Pink JJ Planchon SM Byers KL Bornmann WG Boothman DA Induction of apoptosis in MCF-7WS8 breast cancer cells by beta-lapachone Cancer Res 1998 58 1876 1885 9581828 Robertson N Haigh A Adams GE Stratford IJ Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia Eur Evr J Cancer 1994 30A 1013 1019 10.1016/0959-8049(94)90134-1 Cadenas E Antioxidant and prooxidant functions of DT-diaphorase in quinone metabolism Biochem Pharmacol 1995 49 127 140 7530954 10.1016/S0006-2952(94)00333-5 Ross D Beall H Traver RD Siegel D Phillips RM Gibson NW Bioactivation of quinones by DT-diaphorase, molecular, biochemical, and chemical studies Oncol Res 1994 6 493 500 7620217 Pink JJ Wuerzberger-Davis S Tagliarino C Planchon SM Xiao He Yang Froelich CJ Booth DA Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during β-lapachone-mediated apoptosis Exp Cell Res 2000 255 144 155 10694431 10.1006/excr.1999.4790 Pink JJ Planchon SM Tagliarino C Varnes ME Siegel D Boothman DA NAD(P)H:quinone oxidoreductase activity is the principal determinant of β-lapachone cytotoxicity J Biol Chem 2000 275 5416 5424 10681517 10.1074/jbc.275.8.5416 Boothman DA Trask DK Pardee AB Inhibition of potentially lethal DNA damage repair in human tumor cells by beta-lapachone, an activator of topoisomerase I Cancer Res 1989 49 605 612 2535961 Boorstein RJ Pardee AB β-Lapachone greatly enhances MMS lethality to human fibroblasts Biochem Biophys Res Commun 1984 118 828 834 6704108 Li CJ Zhang LJ Dezube BJ Crumpacker CS Pardee AB Three inhibitors of type 1 human immunodeficiency virus long terminal repeat-directed gene expression and virus replication Proc Natl Acad Sci USA 1993 90 1839 1842 8446597 Li CJ Averboukh L Pardee AB β-lapachone; a novel DNA topoisomerase I inhibitor with a mode of action different from camptothecin J Biol Chem 1993 268 22463 8226754 Frydman B Marton LJ Sun JS Induction of DNA topoisomerase II-mediated DNA cleavage by β-lapachone and related naphthoquinones Cancer Res 1997 57 620 626 9044837 Don MJ Chang YH Chen KK Ho LK Chau YP Induction of CDK inhibitors (p21WAF1 and p27Kip1) and Bak in the β-lapachone-induced apoptosis of human prostate cancer cells Mol Pharmacol 2001 59 784 11259623 Planchon SM Pink JJ Tagliarino C Bornman WG Varnes ME Boothman DA β-Lapachone-induced apoptosis in human prostate cancer cells: Involvement of NQO1/xip3 Experimental Cell Research 2001 267 95 06 11412042 Zhang G Guru V Kain SR Yan G Early detection of apoptosis using a fluorescent conjugate of annexin V Bio Techniques 1997 23 525 531 Cotter TG Martin SJ Techniques in apoptosis; a user's guide 1994 Portland Press Ltd London 10 12 Kumi-Diaka J Rodriguez R Gaudazi G Influence of Genistein (4',5',7-trihydroxyiso-flavone) on the growth and proliferation of selected testicular cells Biology of the Cell 1998 90 349 354 9800352 10.1016/S0248-4900(98)80015-4 Kumi-Diaka J Butler A Caspase-protease activation during the process of genistein-induced apoptosis in TM4 testicular cells Biol Cell 2000 92 115 124 10879632 10.1016/S0248-4900(00)89019-X Kumi-Diaka J Sanderson AA Hall A The mediating role of caspase-3 protease in the intracellular mechanism of genistein-induced apoptosis in human prostatic carcinoma cell lines, DU145 and LNCaP Biology of the Cell 2000 92 595 604 11374438 10.1016/S0248-4900(00)01109-6 Tagliarino C Pink JJ Reinicke KE Simmers SM Wuerzberger-Davis SM Boothman DA Mucalpain activation in β-Lapachone-mediated apoptosis Cancer Biol Ther 2003 2 153 154 12750553

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-591529401910.1186/1477-7827-2-59ResearchExpression and importance of matrix metalloproteinase 2 and 9 (MMP-2 and -9) in human trophoblast invasion Staun-Ram Elsebeth [email protected] Shlomit [email protected] Diana [email protected] Eliezer [email protected] Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Medical Center, 18101, Afula, Israel2 Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel2004 4 8 2004 2 59 59 6 5 2004 4 8 2004 Copyright © 2004 Staun-Ram et al; licensee BioMed Central Ltd.2004Staun-Ram et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The aim of this study was to examine the invasiveness of first trimester trophoblasts according to the secretion profile of MMP-2 and -9 at different gestational stages, and to test the similarity between primary trophoblast cell-culture and the JAR choriocarcinoma cell-line. Methods First trimester trophoblasts were divided into two groups: 6–8 weeks (early) and 9–12 w (late) of gestation. The two trophoblast groups and JAR cells were cultured in medium, with various concentrations of forskolin and Epidermal Growth Factor (EGF). Proteolytic activity was detected by zymography and invasiveness was assessed by Matrigel invasion assay. Student's T-test was used for statistical analysis. Results In 6–8 w trophoblast, proMMP-2 was only slightly dominant over proMMP-9 (53.2% vs. 46.8% respectively), whereas in 9–12 w, proMMP-9 was clearly dominant over proMMP-2 (61.7% vs.38.3% respectively). In JAR cells proMMP-2 was strongly dominant (90.2% vs.9.8% respectively). In JAR cells forskolin significantly increased proMMP-2 and -9 secretion (128.5% +/- 12 and 183.2% +/- 27.9 of control, respectively). EGF had a dual effect on JAR cells: at 8 ng/ml both proMMP-2 and -9 were increased (133.5% +/-15 and 223.9% +/- 32.4 of control, respectively) while at 80 ng/ml both proMMP-2 and -9 were decreased (65.1% +/- 18.3 and 66.6% +/- 37 of control, respectively). Forskolin significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts (125.9% +/- 6.3,128.4% +/- 6.4; 169.7% +/- 20.3, 120.3% +/- 4.5 of control, respectively). EGF also significantly increased both proMMP-2 and -9 secretion in 6–8 w and 9–12 w trophoblasts (141.22% +/- 14.8, 138.8% +/- 10.3; 168.3% +/- 18.2, 117.3 +/- 3.8 of control, respectively). Both forskolin and EGF increased trophoblast cells invasiveness in all groups. The invasive ability of trophoblast cells, induced by forskolin, was reduced by MMP-2 antibody in: JAR cells, 6–8 w and 9–12 w trophoblasts. Likewise trophoblast invasion induced by EGF was reduced by MMP-2 antibody in all groups. However the invasive ability induced by forskolin or EGF was inhibited by MMP-9 antibody only in trophoblasts from 9–12 w. Conclusions First trimester trophoblasts express differential gelatinase secretion profile according to the gestational week. In JAR and early trophoblasts (6–8 w) MMP-2 is the main gelatinase and the key enzyme in trophoblast invasion. Thereafter in late first trimester trophoblasts (9–12 w), both MMP-2 and -9 participate in trophoblast invasion. ==== Body Background Successful implantation depends on the ability of the embryo to degrade the basement membrane of the uterine epithelium and to invade the uterine stroma. Cytotrophoblastic cells (CTB) are derived from trophoectodermal cells of the blastocyst. CTB ensue to become either the villous cytotrophoblastic cells which will proliferate and differentiate by fusion to form the syncytiotrophoblast, or they will stream out of the syncytiotrophoblast to form mononuclear multilayered invasive extravillous cytotrophoblastic cells. The temporal and spatial regulation of trophoblast invasion is mediated in an auto-and paracrinic way by trophoblastic and uterine factors [1]. Several factors have been studied, including hormones, cytokines and growth factors [2,3]. Trophoblast invasion is facilitated by degradation of the extracellular matrix of the endometrium/decidua by various proteinases, among them, the matrix metalloproteinases (MMPs). The tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of the MMPs by binding to the highly conserved zinc-binding site of active MMP [4]. Successful implantation and trophoblast invasion are closely linked to the expression of MMPs, which are able to degrade basement membranes. The gelatinases (gelatinase A: MMP-2: 72-kDa and gelatinase B: MMP-9: 92-kDa) which degrade collagen IV, the main component of the basement membrane, are expressed by trophoblast cells and are therefore regarded as key enzymes in the invasion process [5]. Several studies have shown that MMP-2 and MMP-9 synthesis and activation are required for trophoblast invasion [1,5-9]. Some studies have found either MMP-9 [5,7,8], or MMP-2 [9-11] to be more pronounced during the first stage of trophoblast invasion. However, the exact changes in protease expression during the first trimester are still not clear. Xu et al [11] found differential expression of MMP-2 and -9 in first trimester trophoblast cells, with MMP-2 being the main gelatinase secreted until 9 w and hereafter MMP-9. In this study, trophoblast cells from first trimester were therefore divided into two groups according to their MMP secretion profile. The aim of this study was to examine the expression and importance of MMP-2 and -9 in human trophoblast invasion, and to test the similarity between primary trophoblast cell culture and the JAR choriocarcinoma cell-line. The JAR cell-line serves as a widely used model for 1st trimester trophoblasts [12-14]. The limited availability of 1st trimester trophoblast tissue often requires the use of such a model, and therefore a comparison study between JAR cell-line and 1st trimester trophoblasts is of significant importance to ensure similarity. This study shows a differential, dynamic importance of each gelatinase in trophoblast invasion during the 1st trimester. Methods Cell culture The JAR (Jar, HTB 144) human choriocarcinoma line was established from a trophoblast tumor of the placenta (1988 American Type Culture Collection Catalogue). The JAR cells were a generous gift from Prof. Hochberg A Department of Biological Chemistry, Hebrew University, Jerusalem, Israel. JAR cells (1 × 104 cells/well) were cultured in M-199 medium (Beit-Ha'Emek, Israel) containing 10% Fetal Calf Serum (FCS, Beit-Ha'Emek, Israel) and penicillin/streptomycin (Beit-Ha'Emek, Israel). Cell culture was maintained in a humidified atmosphere containing 5% CO2 at 37°C. After 24 hours of culture to facilitate cell attachment, the medium was removed, and M-199 medium with 1.5% serum supplemented with antibiotics was added. The cells were cultured with various concentrations of: a) Forskolin 1–100 μM, b) Epidermal Growth Factor (EGF), 0.8–80 ng/ml (Sigma). Control consisted of M-199 with 1.5% serum alone. Cells were cultured for an additional 48 hours, and media were removed for analysis of MMP secretion and stored at -20°C until use. Cell count was performed with XTT in order to normalize MMP secretion to cell number. Isolation and cultivation of human cytotrophoblast cells Human trophoblast cells were obtained from legal abortions (6 to 12 weeks of gestational age), with the approval of the local ethical committee (in compliance with the Helsinki Declaration) and the consent of the participating patients. Trophoblast cells were isolated as described previously in detail elsewhere [8,9,15,16] with modifications. Briefly, tissues were digested by 0.25% trypsin (Sigma) and DNase I (Sigma) at 37°C, then trophoblast cells were separated from blood cells and decidua on a discontinuous Percoll gradient (Sigma) and immunopurified with magnetic antibody CD45RB (DAKO, Denmark). The cells were plated at 1–2 × 105 cells/well in 96-well plates or in Transwell plates (Corning) with M-199 medium supplemented with 1.5% FCS and 1% penicillin/streptomycin and kept in 5% CO2 at 37°C. Inducers (10 μM forskolin or 8 ng/ml EGF (chosen as working concentrations after a dose-response study in JAR cell-line) were added to medium, and after 48–72 h media were collected for analysis of MMP secretion and cell count was performed. This method supplies a 95–98% purity of trophoblastic cells, including all trophoblastic sub-groups. We verified the purity of trophoblast cells by using immunohistochemistry with specific antibodies to cytokeratin 7 (positive) and vimentin (negative), commonly used for indication of trophoblast purity [7,8,15]. Figure 1 shows representative pictures of this analysis. Figure 1 Representative immunohistochemical analysis of isolated placental cells (6–8 w) after 24 h in culture. (A) Cells stain positive for anti-human cytokeratin 7 (1:100, Biogenics). (B) Cells stain negative for anti-human vimentin (1:200, Zymed). Magnification: ×100. Cell count assay Evaluation of cell proliferation was performed with XTT Reagent kit (XTT, cell proliferation kit, Beit-Ha'Emek, Israel) according to manufactures protocol. This is based on the activity of mitochondria enzymes in live cells, reducing tetrazolium salts, XTT, into colored formazan compounds, which can be detected colorimetric with a spectrophotometer at 450 nm (ELISA reader). Dye absorbance is proportional to the number of cells in each well. Substrate-gel-electrophoresis (zymography) In order to detect proteolytic activity in conditioned media (CM) collected after 48–72 h culture, substrate-gel-electrophoresis (Zymography) on gels containing gelatin as the substrate were used as was described by our previous manuscript [4]. Briefly, CM, was diluted in sample buffer (5% sodium dodecyl sulphate (SDS), 20% glycerol in 0.4 mol/l Tris, pH 6.8 containing 0.02% Bromophenol Blue without 2-mercaptoethanol) and electrophoresed, through a 10% polyacrylamid gel containing 0.5% gelatin (50 mg/ml). Afterwards gels were washed twice in 2.5% Triton X-100 for 15 min. and incubated for 24 h at 37°C in 0.2 mol/l NaCl, 5 mmol/l CaCl2, 0.2% Brij 35 and 50 mmol/l Tris, pH 7.5. The buffer was decanted and the gels stained with Coomassie Blue G in 30% methanol and 10% acetic acid for 10 min at room temperature on a rotary shaker. Stain was washed out with water until clear bands were seen. Areas where proteolytic activity degraded the gelatin were seen as absence of staining. Identification of each gelatinase band was done in accordance to their molecular weight and commercial standards (gelatinize A and B, 7 μl; Oncogene Science, Cambridge, MA, data not shown). These bands (proMMP) were quantified using the BioImaging gel documentation system (Dinco & Renum, Jerusalem, Israel) endowed with TINA software (Raytest, Staubenhardt, Germany). MMP secretion was expressed as percent of control. Matrigel invasion assay Matrigel invasion assay was prepared in our laboratory with modifications as described in detail elsewhere [17,18]. Briefly, diluted 1:10 Matrigel (1 mg/ml) (BD Biosciences, Beit-Ha'Emek, Israel) in serum free cell culture media was added to upper chamber of 24-well transwell plate, and incubated at 37°C 3–4 h for gelling. JAR Cells were harvested from tissue culture flasks by Trypsin/EDTA, washed and resuspended in 1.5% FCS in M-199 medium and added to upper wells at a density of 105 cells/well in 200 μl medium, while 500 μl medium was added to lower well. 1st trimester trophoblast were cultured in upper wells at a density of 2 × 105 cells/well in 100 μl medium. The same density of cells, in the absence or presence of activators, was seeded in a well without transwell and counted at time of the invasion assay, as reference of total cells. Preliminary studies found no significant matrigel-mediated changes in multiplication rates between 6–8 w and 9–12 w trophoblasts, whether seeded on matrigel or at plastic bottom of well (data not shown). Activators (10 μM Forskolin or 8 ng/ml EGF) and inhibiting MMP-2 or MMP-9 antibodies (Oncogene Cat. IM33L, Cat. IM09L; concentration as recommended by manufacture) were added to medium in upper and lower wells. Plates were incubated at 37°C for 36–48 hours, and then non-invaded cells on top of the transwell were scraped off with a cotton swab. The amount of invaded cells in the lower well as a percent of total seeded cells was evaluated with XTT Reagent kit. The percent of invasion was calculated as: Invasion was expressed as Invasion Index (Percent of control). Statistical methods Results are expressed as mean ± SEM of 5–10 independent experiments, each treatment performed in duplicates. Statistical analysis was performed using the SPSS statistical software. Student's t-test and "one way analysis of variance" (ANOVA) were used when appropriate. P < 0.05 was considered significant. Immunohistochemistry Immunohistochemistry was performed as previously described [19] using the Histostain-Plus kit (Zymed laboratories Inc., USA). Briefly, cultured cells were fixed with cytospray for 20 min and quenched with 3% hydrogen peroxidase in methanol to eliminate endogenous peroxidase activity. The slides were washed, blocked and incubated at room temperature with primary antibodies (mouse anti-human cytokeratin-7 (1:100, clone OVTL12/30, Biogenics) and mouse anti-human vimentin (1:200, clone V9, Zymed laboratories Inc., USA). Secondary antibody used: Histostain-Plus broad-spectrum biotinolated second antibody (Zymed laboratories Inc., USA). Slides were then developed with a substrate-chromagen solution of aminoethyl carbazole (Zymed laboratories Inc., USA). Results Relative secretion profile of proMMP-2 and proMMP-9 in 6–8 W, 9–12 W trophoblasts and in JAR cells without treatment JAR cells (1 × 104 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours, then media collected and gelatinase secretion analyzed by zymography. Figure 2 summarizes the results. In 6–8 w trophoblast, proMMP-2 secretion was only slightly dominant (statistically not significant) compared to proMMP-9, 53.2% vs. 46.8% respectively (SEM ± 4.3). In 9–12 w trophoblasts the picture was different, with proMMP-9 being dominant (P < 0.05) over proMMP-2, 61.7% vs.38.3% respectively (SEM ± 4.6). In JAR cells proMMP-2 was dominant (P < 0.05), whereas proMMP-9 only had a small contribution to the gelatinase secretion, 90.2% vs.9.8% respectively (SEM ± 1.4). Figure 2 (A) Representative secretion pattern of proMMP-2 (72 kD) and proMMP-9 (92 kD) in 6–8 w, 9–12 w trophoblasts and in JAR cells without treatment as examined with zymography. (B) Bar graph describing the relative percentage of gelatinases secretion, representing mean ± SEM from 5 independent experiments. Black bars represents proMMP-2 and white bars represent proMMP-9, *P < 0.05. Dose-dependent effect of forskolin and EGF on the proMMP-2 and -9 secretion by JAR cells JAR cells (1 × 104 cells/well) were incubated 48 hours in the absence or presence of forskolin (1 μM, 10 μM or 100 μM) or EGF (0.8 ng/ml, 8 ng/ml or 80 ng/ml) and media was analyzed by zymography for gelatinase secretion. Fig 3 summarizes the results. 10 μM forskolin significantly enhanced secretion of proMMP-2 compared to control (128.5% ± 12.0, P < 0.05). Forskolin (1 μM and 10 μM) significantly enhanced proMMP-9 secretion compared to control (131.3% ± 11.1, and 183.2% ± 27.9, P < 0.05, respectively) (Fig. 3A). 8 ng/ml EGF significantly enhanced secretion of proMMP-2 (133.5% ± 15.0, P < 0.05) and of proMMP-9 (223.9% ± 30.4, P < 0.05) compared to control. 80 ng/ml EGF, on the contrary, decreased proMMP-2 and proMMP-9 secretion compared to control (65.1% ± 18.3, and 66.6% ± 3.7, P < 0.05) (Fig. 3B). Figure 3 Dose-dependent effect of Forskolin and EGF on gelatinase secretion in JAR cells. JAR cell (1 × 104/well) were incubated 48 hours with or without Forskolin (1 μM, 10 μM or 100 μM) or EGF (0.8 ng/ml, 8 ng/ml or 80 ng/ml) and media collected for measurement of Gelatinase secretion. (A) Bar graph, representing mean ± SEM of 10 independent experiments, of cells treated with forskolin. (B) Bar graph, representing mean ± SEM of 10 independent experiments, of cells treated with EGF. Black bars represent proMMP-2 and gray bars represent proMMP-9. *P < 0.05 vs. control. Effect of forskolin on proMMP-2 and proMMP-9 secretion by JAR cells, 1st trimester trophoblast cells 6–8 w and 9–12 w of gestation JAR cells (1 × 104 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours in the absence or presence of forskolin 10 μM. Figure 4A shows representative zymography gels. Figure 4B and 4C summarizes the results. Gelatinase secretion was enhanced by forskolin in all cell groups: Forskolin significantly increased proMMP-2 secretion in JAR cells (144.3% ± 8.8, P < 0.05), in 6–8 w trophoblast (125.9% ± 6.3, P < 0.05) and in 9–12 w trophoblast (169.7% ± 20.3, P < 0.05) as compared to control (Fig. 4B). Forskolin also significantly increased proMMP-9 secretion in JAR cells (226.6% ± 50.7, P < 0.05), in 6–8 w trophoblast (128.4% ± 6.4, P < 0.05), and in 9–12 w trophoblast (120.3% ± 4.5, P < 0.05) as compared to control (Fig. 4C). Figure 4 Secretion of proMMP-2 and proMMP-9 after 48 hours incubation of JAR cells, 1st trimester trophoblast cells 6–8 week or 1st trimester trophoblast cells 9–12 week in medium in absence or presence of 10 μM Forskolin. (A) Representative zymography gels. (B) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-2 (72 kD). (C) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-9 (92 kD). White bars represent control medium of cells without treatment. Black bars represent medium from cells with forskolin treatment. *P < 0.05 vs. control. Effect of EGF on proMMP-2 and proMMP-9 secretion by JAR cells, 1st trimester trophoblast cells 6–8 w and 9–12 w of gestation JAR cells (1 × 104 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (1–2 × 105 cells/well) were incubated for 48 hours in the absence or presence of EGF 8 ng/ml. Figure 5A shows representative zymography gels. Figure 5B and 5C summarizes the results. EGF significantly increased proMMP-2 secretion in JAR cells (130.4% ± 13.1, p < 0.05), in 6–8 w trophoblast (141.22% ± 14.8, P < 0.05) and also in 9–12 w trophoblast (168.3% ± 18.2, P < 0.05) as compared to control (Fig. 5B). EGF significantly increased proMMP-9 secretion in JAR cells (187.8% ± 27.3, P < 0.05), in 6–8 w trophoblast (138.8% ± 10.3, P < 0.005), and in 9–12 w trophoblast (117.3% ± 3.8, P < 0.05) as compared to control (Fig. 5C). Figure 5 Secretion of proMMP-2 and proMMP-9 after 48 hours incubation of JAR cells, 1st trimester trophoblast cells 6–8 week or 1st trimester trophoblast cells 9–12 week in medium in absence or presence of 8 ng/ml EGF. (A) Representative zymography gels. (B) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-2 (72 kD). (C) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-9 (92 kD). White bars represent control medium of cells without treatment, black bars represent medium from cells with EGF treatment. *P < 0.05 vs. control. Effect of forskolin on cell invasion properties in JAR cells, 1st trimester trophoblast cells 6–8 w and 9–12 w of gestation JAR cells (105 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (2 × 105 cells/well) were incubated for 36–48 hours in the absence or presence of forskolin 10 μM on top of Transwell wells containing a transwell membrane coated with matrigel. Forskolin (10 μM) significantly enhanced trophoblast invasion in all cell groups. Forskolin increased cell invasion in JAR cells (110.6% ± 3.4, P < 0.05) (Fig. 6A), in 6–8 w 1st trimester trophoblast (189.7% ± 14.2, P < 0.05) (Fig. 6B) and in 9–12 w (302.4% ± 56.0, P < 0.05) as compared to control (Fig. 6C). The addition of inhibitory MMP-2 antibody significantly decreased invasion of control cells of JAR cells (86.5% ± 3.6, P < 0.05) and of 6–8 w (73.8 ± 10.6, P < 0.05) but did not affect 9–12 w trophoblasts. In forskolin-induced cells the presence of inhibitory MMP-2 antibody caused a significant decrease in invasion of JAR cells compared with induced cells alone (96.6% ± 1.5 versus 110.6% ± 3.4, respectively P < 0.05), of 6–8 w trophoblasts compared with induced cells alone (114.1% ± 24.6 versus 189.7% ± 14.2, respectively, P < 0.05) and of 9–12 w trophoblasts compared with induced cells alone (188.8% ± 18.4 versus 302.4% ± 56.0, respectively, P < 0.05) (Figure. 6A,6B and 6C) Figure 6 Cell invasion ability of JAR cells, 1st trimester trophoblast cells 6–8 week or 1st trimester trophoblast cells 9–12 week tested with Transwell Invasion Assay. (A) represents JAR cells, (B) 1st trimester trophoblast cells 6–8 week and (C) 1st trimester trophoblast cells 9–12 week. Cells were treated with 10 μM Forskolin and incubated 36 hours on Matrigel coated membrane, with or without MMP-2 or MMP-9 inhibitory antibodies. Cells that invaded the membrane to lower well were counted with XTT. Results represent mean ± SEM from 10 independent experiments. Black bars represent control (cells without treatment or cells treated with Forskolin) with no antibodies. Gray bars represent cells (without treatment or treated with Forskolin) with addition of MMP-2 inhibitory antibody. White bars represent cells (without treatment or treated with Forskolin) with addition of MMP-9 inhibitory antibody. ANOVA for all groups results in p < 0.05, post test confirmed the t test results. *P < 0.05 The addition of inhibitory MMP-9 antibody surprisingly increased invasion of control cells of 6–8 w and 9–12 w trophoblast (132.0 ± 8.3 and 134.9 ± 15.2, respectively, P < 0.05) as compared to control, and only in 9–12 w forskolin-induced cells caused a significant decreased invasion compared with induced cells alone (201.5% ± 6.3 versus 302.4% ± 56.0, respectively, P < 0.05). ANOVA post test confirmed the t test results (Fig. 6A,6B and 6C). Effect of EGF on cell invasion properties in JAR cells, 1st trimester trophoblast cells 6–8 w and 9–12 w of gestation JAR cells (105 cells/well), 1st trimester trophoblast cells 6–8 w and 9–12 w (2 × 105 cells/well) were incubated for 36–48 hours in the absence or presence of EGF 8 ng/ml on top of Transwell wells containing a transwell membrane coated with matrigel. The results of EGF resembled those of forskolin. EGF enhanced trophoblast invasion in all cell groups. EGF increased cell invasion in JAR cells (112.6% ± 2.9, P < 0.05) (Fig. 7A), in 6–8 w 1st trimester trophoblast (157.9% ± 10.4, P < 0.05) (Fig. 7B) and in 9–12 w (192.4% ± 10.5, P < 0.05) compared to control (Fig. 7C). In EGF-induced cells the presence of inhibitory MMP-2 antibody caused a significant decrease in invasion of JAR cells (100.2% ± 0.8 versus 112.6 ± 2.9, P < 0.05), of 6–8 w trophoblasts (129.7% ± 8.0 versus 157.9 ± 10.4, P < 0.05) and of 9–12 w trophoblasts (161.1% ± 22.0 versus 192.4 ± 10.5, P < 0.05) compared with induced cells alone (Figure. 7A,7B and 7C). Figure 7 Cell invasion ability of JAR cells, 1st trimester trophoblast cells 6–8 week or 1st trimester trophoblast cells 9–12 week tested with Transwell Invasion Assay. (A) represents JAR cells, (B) 1st trimester trophoblast cells 6–8 week and (C) 1st trimester trophoblast cells 9–12 week. Cells were treated with 8 ng/ml EGF and incubated 36 hours on Matrigel coated membrane, with or without MMP-2 or MMP-9 inhibitory antibodies. Cells that invaded the membrane to lower well were counted with XTT. Results represent mean +SEM from 10 independent experiments. Black bars represent control (cells without treatment or cells treated with EGF) with no antibodies. Gray bars represent cells (without treatment or EGF treated) with addition of MMP-2 inhibitory antibody. White bars represent cells (without treatment or EGF treated) with addition of MMP-9 inhibitory antibody. ANOVA for all groups results in p < 0.05, post test confirmed the t test results. * P < 0.05. The addition of inhibitory MMP-9 antibody affected 9–12 w EGF–induced cells and caused a significant decreased invasion compared with induced cells alone (169.7% ± 18.1 versus 192.4 ± 10.5, respectively, P < 0.05), ANOVA post test confirmed the t test results (Figure. 7A,7B and 7C). The inhibitory affect of MMP-2/-9 antibody on control cells was described in the previous section. Discussion Trophoblastic invasion of the endometrium is highly regulated by interrelated reactions between invasion-promoting factors, such as cytokines, growth factors, MMP-2 and MMP-9, and invasion-inhibiting factors such as TIMPs. In the current study, the secretion and activity of MMP-2 and MMP-9 in human cytotrophoblastic cells from different weeks of gestation was measured and compared with a choriocarcinoma cell-line. A differential secretion profile of proMMP-2 and -9 was found between 6–8 w and 9–12 w as expressed by a shift in the relative proportion of each gelatinase. Elevated proMMP-9 secretion in 9–12 w was observed, compared with both 6–8 w and JAR cells. These results are consistent with those of Xu et al [11], who found MMP-2 production to be dominant between 6–8 weeks of gestation and then declining, whereas MMP-9 production significantly increased from 8 to 11 weeks, with a shift of dominant gelatinase from MMP-2 to MMP-9 from 9 week of gestation. MMP-2 is predominant in human preimplantation embryos [20,21], whereas MMP-9 is dominant in the third trimester [6]. Niu et al [22] reported a dominance of MMP-2 secretion over MMP-9 from 1st trimester villous tissue, and a dramatically decrease in MMP-2 levels in the second trimester. These results support our findings of a dynamic gelatinase secretion profile during the 1st trimester. In our study, MMP secretion was induced by two separate signal pathways: PKA and PTK. Several factors with importance in embryo implantation act via the cAMP-protein kinase A (PKA) signal transduction pathway, including human chorion gonadotropin (hCG), the primary signal of an implanting pregnancy [23]. Forskolin is a prototypical stimulator of the cAMP pathway by direct activation of adenylate cyclase [24]. In this study, forskolin was found to significantly enhance proMMP-2 secretion in JAR choriocarcinoma cell-line, 6–8 w and 9–12 w trophoblasts. Forskolin also enhanced proMMP-9 secretion, in choriocarcinoma cells and in 1st trimester trophoblast (from both groups). This indicates that forskolin might influence trophoblast cells invasiveness by enhancing the secretion of gelatinases. Zymography measures all forms of MMP (active and inactive) and therefore does not reliably represent the true physiological activity, which is influenced by the presence of MMP inhibitors and activators. In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PKA stimulation in JAR cells, 6–8 and 9–12 w trophoblasts. Our results showed a significant increase in invasive ability in JAR cells and in early and late 1st trimester trophoblasts, after stimulation with forskolin. In order to detect the contribution of each gelatinase to this invasive process, inhibitory antibodies to MMP-2 and MMP-9 were added to cell culture and cell invasive ability examined. MMP-2 inhibitory antibody caused a significant decrease in cell invasion in JAR cells, 6–8 w and 9–12 w trophoblasts treated with forskolin, whereas MMP-9 inhibitory antibody only caused a decrease in 9–12 w trophoblasts. This indicates, that most probably MMP-2 and not MMP-9 is the key-enzyme in the invasion process of JAR cells and early 1st trimester trophoblasts stimulated by forskolin, whereas MMP-9 together with MMP-2 plays a role in late 1st trimester trophoblasts. Only a few published data describe the relationship between forskolin and trophoblast invasion. Human chorionic gonadotropin acts via cAMP, and is considered a sign of differentiation of trophoblasts to syncytiotrophoblast. The hCG receptor was shown to be expressed on invasive trophoblast and in choriocarcinoma cells, and hCG was found to increase in vitro invasion and migration of a trophoblastic cell line, an effect that was also mimicked by forskolin [23]. Several studies demonstrated a positive correlation between hCG level and successful implantation [25] or inappropriate implantation/ invasion associated with the development of preeclampsia [24] or gestational trophoblastic tumors [27]. To the best of our knowledge, our study is the first to report the effect of forskolin on MMPs in first trimester trophoblastic cells. In addition, this study also distinguishes between the contributions of each of the gelatinases to the invasive capacity enhanced by forskolin in trophoblastic cells. Epidermal Growth factor (EGF) plays a major role in placental implantation, growth and differentiation and is regarded a paracrinic factor modifying the implantation process. EGF acts on trophoblasts via a specific receptor (EGFR) from the tyrosine kinase receptor family [28]. EGF is secreted from the endometrium during the implantation window, in which the embryo also expresses EGFR [29], and expressed in placenta from 1st throughout third trimester [30] In this study EGF stimulated secretion of proMMP-2 in JAR cells, 6–8 w and 9–12 w trophoblasts and also enhanced the secretion of proMMP-9 in JAR, in 6–8 w trophoblasts and in 9–12 w trophoblasts. Our results therefore indicate, that MMP-2 and MMP-9 secretion by trophoblastic cells may be stimulated through the PTK pathways during the first trimester. We noted that EGF at a high concentration (80 ng/ml), in contrast, decreased proMMP-2 and -9 secretion in JAR cells. This result corresponds with previous published data regarding a dual, concentration dependent effect of EGF on cell functions [31,32]. In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PTK stimulation in JAR cells, 6–8 and 9–12 w trophoblasts. Our results showed a significant increase in invasion ability in JAR cells and in early and late 1st trimester trophoblasts, after stimulation with EGF. In EGF-stimulated JAR cells and in 6–8 w and 9–12 w trophoblasts inhibitory MMP-2 antibody decreased cell invasion, whereas inhibitory MMP-9 antibody caused a significant decrease in invasion only in 9–12 w trophoblasts. We thereby showed, that in EGF stimulated cells as well, MMP-2 is the key-enzyme in the invasion process in vitro in JAR cell and in early 1st trimester trophoblasts, whereas in late 1st trimester trophoblasts both MMP-2 and MMP-9 have a role. EGF was found to induce changes in morphology and to increase invasive capacity of first trimester trophoblasts, whereas later gestational cytotrophoblasts (2nd trimester), whose invasive capacity is diminished, are much less affected [29]. EGF was also found to increase MMP-9 secretion by cytotrophoblasts [33]. In JAR control cells inhibitory, MMP-2 antibody also decreased invasion, whereas MMP-9 antibody had no affect, indicating that the basic invasive ability of these choriocarcinoma cells is mainly due to MMP-2 and not to MMP-9. Inhibition of MMP-2 in control cells also decreased invasion in 6–8 w trophoblast, but not in 9–12 w, indicating again the importance of MMP-2 in early (6–8 w) trophoblast invasiveness. Surprisingly inhibition of MMP-9 in 6–8 w and 9–12 w trophoblasts without treatment caused an increase in invasion. We speculate that this may be due to a release of other proteinases, including other MMPs, since MMP-9 is known to dimerize [34,35] All in all we found, that in early 1st trimester trophoblasts (6–8 w), MMP-2 is the major gelatinase participant in cell invasion, whereas in later 1st trimester trophoblasts (>9 w) both MMP-9 and MMP-2 most probably participate in cell invasion, however we cannot exclude the possibility of other MMP family members participating in this process. Isaka et al [9] have shown that invasive ability of early first trimester trophoblast was inhibited by MMP-2 antibody in a dose dependent manner, thereby suggesting that the invasive ability of trophoblasts may be regulated by the enzyme activity of gelatinases, especially MMP-2. This study supports ours in the involvement of MMP-2 in trophoblast cell invasion. MMP-2 was found to be located in invasive evCTB in 1st trimester placenta [9,10,36], whereas MMP-9 was located in the non-invasive vCTB [9]. In contrast, several studies have found MMP-9 to be the key-enzyme in trophoblast invasion in vitro [1,5,7]. We speculate, that the main reason for this controversy of results comes from the dynamic gelatinase expression during the 1st trimester, as earlier discussed. The choice of pathway for stimulation of cell invasion may also contribute to a difference in results. We used stimulation through the PKA and PTK pathways; whereas other groups used the PKC pathway [7,8]. Various stimulators, inducing different signal pathways, are likely not to affect the same enzymes in an identical manner, and thereby can result in varying dominant enzymes. We found, that MMP-2 is also the key-enzyme in JAR cell invasion; therefore JAR cells resemble early 1st trimester trophoblasts in cell invasive ability and in MMP secretion profile and differ from late 1st trimester trophoblast in these parameters. It has been documented, that JAR-trophoblast cells have the ability to invade in vivo [37,38]. We chose this cell-line because it provides a large number of uniform cells and preserve the ability to differentiate into syncytiotrophoblast-like cell in vitro [39,40]. Other studies showed different compartment in vitro between choriocarcinoma cell-lines and human first trimester trophoblast in the regulation of invasion [36,37]. The study of JAR cell invasion may therefore represent only partly the aspects and mechanisms of the in vivo situation of invasion, where many cell types are involved. Conclusions We showed that forskolin and EGF stimulate proMMP-2 and -9 secretion from trophoblasts, and that there is a differential, dynamic importance of each gelatinase in trophoblast invasion during 1st trimester. We suggest that MMP-2 is the key-enzyme in JAR and early 1st trimester (6–8 w) trophoblast invasion, whereas both MMP-2 and -9 are important for late (9–12 w) trophoblast invasion. Acknowledgement We thank Desoye G, clinic of Obstetrics and Gynaecology, Austria for generously providing the protocol of trophoblast isolation. ==== Refs Bischop P Campana A A Putative role for oncogenes in trophoblast invasion? Human Reproduction 2000 15 51 58 11261483 Meisser A Chardonnens D Campana A Bischof P Effects of tumor necrosis factor alpha, interleukin-1 alpha, macrophage colony stimulating factor and transforming growth factor beta on trophoblastic matrix metalloproteinases Mol Hum Reprod 1999 5 252 260 10333360 10.1093/molehr/5.3.252 Meisser A Cameo P Islami D Campana A Bischof P Effects of interleukin-6 (IL-6) on cytotrophoblastic cells Mol Hum Reprod 1999 5 1055 1058 10541568 10.1093/molehr/5.11.1055 Goldman S Weiss A Eyali V Shalev E Differential activity of the gelatinases (matrix metalloproteinases 2 and 9) in the fetal membranes and deciduas, associated with labour Molecular Human Reproduction 2003 9 367 373 12771238 10.1093/molehr/gag040 Librach CL Werb Z Fitzgerald ML Chiu K Corwin NM Esteves RA Grobelny D Galardy R Damsky CH 92-kDa type IV collagenase mediates invasion of human cytotrophoblasts J Cell Biol 1991 113 437 449 1849141 10.1083/jcb.113.2.437 Shimonovitz S Hurwitz A Dushnik M Anteby E Geva-Eldar T Yagel S Developmental regulation of the expression of 72 and 92 kDa type collagenases in human trophoblasts: a possible mechanism for control of trophoblast invasion Am J Obstet Gynecol 1994 171 832 838 7522400 Bischof P Maelli M Campana A Importance of matrix metalloproteinases (MMP) in human trophoblast invasion Early Pregnancy 1995 1 263 269 9363258 Bischof P Truong K Campana A Regulation of Trophoblastic Gelatinases by Proto-oncogenes Placenta 2003 24 155 163 12566242 10.1053/plac.2002.0890 Isaka K Usuda S Ito H Sagawa Y Nakamura H Nishi H Suzuki Y Li F Takayama M Expression and activation of Matrix Metalloproteinase 2 and 9 in Human Trophoblasts Placenta 2003 24 53 64 12495660 10.1053/plac.2002.0867 Bjoern SF Hastrup N Larsen JF Lund LR Pyke C Messenger RNA for Membrane-type Matrix Metalloproteinase, MT2-MMP, is expressed in Human Placenta of first Trimester Placenta 2000 21 170 176 10736239 10.1053/plac.1999.0447 Xu P Wand Y Zhu S Luo S Piao Y Zhuang L Expression of Matrix Metalloproteinase-2,-9, and -14, Tissue Inhibitors of Metalloproteinases-1, and Matrix proteins in Human Placenta During the First Trimester Biology of Reproduction 2000 62 988 994 10727268 Moriyama T Otani T Maruo T Expression of adrenomedullin by human placental cytotrophoblasts and choriocarcinoma JAr cells J Clin Endocrinol Metab 2001 86 3958 3961 11502838 10.1210/jc.86.8.3958 Gude NM Stevenson JL Rogers S Best JD Kalionis B Huisman MA Erwich JJ Timmer A King RG GLUT12 expression in human placenta in first trimester and term Placenta 2003 24 566 570 12744934 10.1053/plac.2002.0925 Bamberger AM Ezzat S Cao B Wong M Parker KL Schulte HM Asa SL Expression of steroidogenic factor-1 (SF-1) mRNA and protein in the human placenta Mol Hum Reprod 1996 2 457 461 9238716 Blaschitz A Weiss U Dohr G Desoye G Antibody Reaction Patterns in First Trimester Placenta: Implications for Trophoblast Isolation and Purity Screening Placenta 2000 21 733 741 10985978 10.1053/plac.2000.0559 Bischof P Friedli E Martelli M Campana A Expression of extracellular matrix-degrading metalloproteinases by cultured human cytotrophoblast cells: effects of cell adhesion and immunopurification Am J Obstet Gynecol 1991 165 1791 1801 1750477 Staff AC Ranheim T Henriksen T Halvorsen B 8-Iso-prostaglandin F2 a reduces trophoblast invasion and matrix metalloproteinase activity Hypertension 2000 35 1307 1313 10856282 Imamura H Takao S Aikou T A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion Cancer Res 1994 54 3620 3624 8012990 Anteby EY Greenfield C Natanson-Yaron S Goldman-Wohl D Hamani Y Khudyak V Yagel S Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase 9 Mol Hum Reprod 2004 10 229 235 14996996 10.1093/molehr/gah031 Puistola U Ronnberg L Martikainen H Turpeenniemi-Hujanen T The human embryo produces basement membrane collagen (type IV collagen)-degrading protease activity Hum Reprod 1989 4 309 311 2715307 Turpeenniemi-Hujanen T Feinberg RF Kauppila A Puistola U Laminin in the human embryo implantation: analog to the invasion by malignant cells Fertil Steril 1992 58 105 113 1378028 Niu R Okamoto T Iwase K Nomura S Mizutani S Quantitative Analysis of Matrix Metalloproteinases-2 and -9, and their Tissue Inhibitors-1 and -2 in Human Placenta throughout Gestation Life Sciences 2000 66 1127 1137 10737363 10.1016/S0024-3205(00)00416-1 Zygmunt M Hahn D Munstedt K Bischof P Lang U Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro Placenta 1998 19 587 593 9859862 10.1016/S0143-4004(98)90019-4 Yamamoto T Matsumoto K Kurachi H Okamoto Y Nishio Y Sakata M Tasaka K Murata Y Progesterone inhibits transcriptional activation of human chorionic gonadotropin-alpha through protein kinase A pathway in trophoblast cells Mol Cell Endocrinol 2001 182 215 224 11514056 10.1016/S0303-7207(01)00580-9 Sunder S Lenton EA Endocrinology of the peri-implantation period Baillieres Best Pract Res Clin Obstet Gynaecol 2000 14 789 800 11023800 10.1053/beog.2000.0119 Bahado-Singh RO Oz AU Kingston JM Shahabi S Hsu CD Cole L The role of hyperglycosylated hCG in trophoblast invasion and the prediction of subsequent pre-eclampsia Prenat Diagn 2002 22 478 481 12116306 10.1002/pd.329 Vuong PN Guillet JL Houissa-Vuong S Lhomme C Proust A Cristalli B Pathology of gestational trophoblastic tumors Gynecol Obstet Fertil 2000 28 913 926 11192198 10.1016/S1297-9589(00)00002-3 Evain-Brion D Alsat E Epidermal growth factor receptor and human fetoplacental development J Pediatr Endocrinol 1994 7 295 302 7735366 Bass KE Morrish D Roth I Bhardwaj D Taylor R Zhou Y Fisher SJ Human cytotrophoblast invasion is up-regulated by Epidermal Growth Factor: Evidence that Paracrine Factors modify This Process Developmental Biology 1994 164 550 561 8045351 10.1006/dbio.1994.1223 Hofmann GE Scott RT JrBergh PA Deligdisch L Immunohistochemical localization of epidermal growth factor in human endometrium, decidua, and placenta J Clin Endocrinol Metab 1991 73 882 887 1890159 Chajry N Martin PM Pages G Cochet C Afdel K Berthois Y Relationship between the MAP kinase and the dual effect of EGF on A431 cell proliferation Biochem Biophys Res Commun 1994 203 984 990 8093084 10.1006/bbrc.1994.2279 Hsieh JS Wang JY Huang TJ The role of epidermal growth factor in gastric epithelial proliferation in portal hypertensive rats exposed to stress Hepatogastroenterology 1999 46 2807 2811 10576350 Sawicki G Radomski MW Winkler-Lowen B Krzymien A Guilbert LJ Polarized Release of Matrix Metalloproteinase-2 and -9 from cultured Human Placental Syncytiotrophoblasts Biology of Reproduction 2000 63 1390 1395 11058543 Winberg JO Kolset SO Berg E Uhlin-Hansen L Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans J Mol Biol 2000 304 669 680 11099388 10.1006/jmbi.2000.4235 Olson MW Bernardo MM Pietila M Gervasi DC Toth M Kotra LP Massova I Mobashery S Fridman R Characterization of the monomeric and dimeric forms of latent and active matrix metalloproteinase-9. Differential rates for activation by stromelysin 1 J Biol Chem 2000 275 2661 2668 10644727 10.1074/jbc.275.4.2661 Nawrocki B Polette M Marchand V Maquoi E Beorchia A Tournier JM Foidart JM Birembaut P Membrane-type matrix metalloproteionase-1 expression at the site of human placentation Placenta 1996 17 565 572 8916204 Yagel S Parhar RS Jeffrey JJ Lala PK Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells J Cell Physiol 1988 136 455 462 3170642 Grummer R Hohn HP Mareel MM Denker HW Adhesion and invasion of three human choriocarcinoma cell lines into human endometrium in a three-dimensional organ culture system Placenta 1994 15 411 429 7937597 Eldar-Geva T Rachmilewitz J de Groot N Hochberg A Interaction between choriocarcinoma cell line (JAr) and human cytotrophoblasts in vitro Placenta 1993 14 217 223 7685096 Hochberg A Rachmilewitz J Eldar-Geva T Salant T Schneider T de Groot N Differentiation of choriocarcinoma cell line (JAr) Cancer Res 1992 52 3713 2717 1617644

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==== Front Nucl ReceptNuclear Receptor1478-1336BioMed Central London 1478-1336-2-51531894210.1186/1478-1336-2-5ResearchBinding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA) Taguchi Yasuto [email protected] Mirek [email protected] Donald L [email protected] Obstetrics and Gynecology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA2 Endocrinology and Metabolism, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia 30033, USA3 Geriatrics, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA2004 19 8 2004 2 5 5 15 7 2003 19 8 2004 Copyright © 2004 Taguchi et al; licensee BioMed Central Ltd.2004Taguchi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA (E2-BSA) has been widely used as an agent to study these novel membrane-associated ER events. However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 (usually between 3–5%), the latter was carefully removed by ultrafiltration. Results As previously reported, a 10-to 1000-fold molar excess of E2-BSA was unable to compete with 3H-E2 binding to ER when added simultaneously. However, when ER was pre-incubated with the same concentrations of E2-BSA, the binding of 3H-E2 was significantly reduced. E2-BSA binding to a putative membrane-associated ER was directly visualized using fluorescein labeled E2-BSA (E2-BSA-FITC). Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780. Conclusions These results demonstrate that E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER. ==== Body Background For many years, estrogen actions have been presumed to be mediated almost exclusively through the regulation of target gene transcription by a chromosomal bound estrogen receptor. These genomic estrogen effects are the well described interactions between the estrogen receptor and adapter transcription factors that result in activation or inhibition of the basal transcription protein machinery. However, there is a growing body of evidence that several rapid estrogen effects are non-transcriptional in nature. These rapid estrogen effects include changes of calcium flux in several cell types [1-3], MAPK activation [4,5], cAMP levels [6,7], and nitric oxide release [8]. That many of these effects are mediated by a membrane-localized estrogen receptor has been postulated for some time [9,10], but the majority of evidence supporting this hypothesis is indirect, relying on the induction of these non-genomic effects using estrogen covalently conjugated to BSA by a 6 atom hydrocarbon tether (E2-BSA) [11,12]. However, the relative binding efficiency of these conjugates is low and concern has been raised regarding the use of these conjugates as direct surrogates for estrogen [13]. A recent report added to this controversy by showing that commercially available E2-BSA is contaminated by unconjugated free E2 and a series of binding experiments demonstrated that E2-BSA was unable to bind to ER after the contaminant E2 was removed. [14]. These findings contradict studies where fluorescein-labeled E2-BSA (E2-BSA-FITC) specifically bound to a putative ER on the cell membrane [15-17]. Elucidation of novel membrane-associated ER effects is crucial to our understanding of the non-genomic signaling pathways of ER and other hormone receptors. Hormone-conjugated BSA is an important tool in this pursuit. We believe the contradictory results are explained by differences in the rates of binding of the bulky E2-BSA and E2 with the ER. We show that pre-incubation of E2-BSA with ERα results in a highly significant decrease in the binding of 3H-E2. The binding of 3H-E2 with ERα is unaffected by the simultaneous addition of E2-BSA. We also demonstrate that fluorescein conjugated E2-BSA binds to the membrane of cells that endogenously produce ERα and to HeLa cell lines stably expressing mERα. Results E2-BSA binding to purified estrogen receptor Although E2 is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E2-BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E2-BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E2-BSA, maximizing the amount of E2-BSA bound to the receptor. To test this hypothesis, E2-BSA free of contaminant E2 was prepared by ultrafiltration. Competition between the purified E2-BSA and labeled E2 for binding to purified ERα was determined after E2-BSA was pre-incubated with ERα and also when added at the same time as labeled E2. As shown in figure 1, concurrent addition of labeled E2 and E2-BSA had no effect on labeled E2 binding. However, a four-hour pre-incubation of E2-BSA with ER significantly decreased E2 binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E2-BSA. Figure 1 Pre-incubation of purified hERα with E2-BSA competes for estradiol binding. Purified ERα was incubated with E2 (solid line) or E2-BSA (dotted line) for four hours before (a) or concurrently with (b) the addition of labeled E2. Incubation was continued for another 2 h at room temperature, and at the end of this period, specific binding was determined by adsorption, removal, and counting of free labeled E2. E2-BSA binding to ER in intact cells Non-genomic actions of the estrogen receptor are now well established. Several investigators have demonstrated that fluorescein labeled E2-BSA (E2-BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E2. Specific binding of E2-BSA-FITC to this membrane-localized form of the ER would further establish that E2 conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E2-BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E2 to HeLa-ERα, but not native HeLa cells (figure 2). Scatchard analysis of the binding of E2 to HeLa-ERα cells showed that although weakly expressed, the Kd for the expressed ERα was 7.04 nM, similar to published values (figure 3). HeLa-ERα cells, but not native HeLa cells, exhibited fluorescent staining of the cell membrane after incubation with E2-BSA-FITC (figure 4). The heterogeneous staining pattern reflected the low level of ERα expression. This fluorescence was not seen when HeLa-ERα cells were incubated with BSA conjugated to fluorescein alone (data not shown). Figure 2 Whole cell binding of estradiol to MC7 cells and HeLa cells stably transfected with ERα. 2 × 106 cells were incubated with 3H-17β-estradiol (10-8 M) in the absence (solid) and presence (white) of 100 fold excess of unlabeled estradiol for 15 minutes at room temperature, washed, and placed on ice for 30 minutes. Cells were then pelleted, lysed and counted. Results are expressed as the mean +/- the SEM of 3 experiments (* p < 0.01) Figure 3 Estradiol binding to HeLa cells stably transfected with ERα. Subconfluent HeLa-ERα cells were trypsinized and aliquots (2 × 106 cells) incubated with several concentrations of 3H-17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. a) Representative binding results of 3 independent experiments with total binding (solid box), non-specific binding (open box), and specific binding (triangle). b) Scatchard analysis of binding results. Figure 4 Membrane localization of ERα. HeLa cells stably transfected with ERα(a) and native HeLa cells (b) were incubated with fluorescein labeled membrane impermeable BSA conjugated to estradiol (E2-BSA-FITC) and visualized under phase contrast bright field and with UV light with an excitation filter for FITC. To establish the specificity of E2-BSA binding, MC7 cells and HeLa-ERα cells were incubated with E2-BSA-FITC after pre-incubation with various concentrations of E2 and the anti-estrogen ICI-182,780. As shown in figure 5, fluorescence was lost in both cell types in a dose dependent manner with increasing concentrations of E2. Fluorescence was almost completely eliminated by pre-incubation with the specific competitor ICI-182,780. BSA conjugated to FITC alone did not bind. These results suggest that estrogen covalently bound to BSA can bind to ER in a biologically significant manner. Figure 5 E2 competition with E2-BSA-FITC binding. MC7 cells (black bars) or HeLa-ERα (white bars) were incubated with vehicle, various concentrations of E2, or ICI 182,780 (10-8 M) for 30 minutes and then incubated an additional 30 minutes with E2-BSA-FITC or BSA-FITC alone (grey bar). Cells were fixed, and visualized by confocal microscopy. Digitized images were inverted to black on white and pixel density for each cell was determined by averaging the density across the cell membrane at four orthogonal points. Each bar represents >20 cells counted +/- SEM. (* p < 0.05). The possibility that E2-BSA-FITC could be degraded during incubation with intact cells was examined using HPLC. E2-BSA-FITC was incubated in empty wells or wells containing MC7 cells under the same conditions employed for the binding studies described above. Media was removed from the cells and HPLC performed with a reverse phase column. Peaks were visualized using a scanning fluorescence detector. Aqueous solutions of E2-BSA-FITC produced a single peak with a retention time of 5.5 minutes using a methanol-water gradient from 80% to 50% over 30 minutes at 1 ml/minute. E2 and E2-BSA did not fluoresce at the excitation and emission wavelengths used (data not shown). Spectra obtained from media containing E2-BSA-FITC alone and media containing E2-BSA-FITC incubated with MC7 cells are shown in figure 6. The average area under the curve for E2-BSA-FITC was the same (p < 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E2-BSA-FITC is stable under the culture conditions employed for the binding experiments. Figure 6 Stability of E2-BSA-FITC. E2-BSA-FITC (10-8 M in estrogen) was placed in empty wells or wells containing MC7 cells and incubated for 30 minutes at 4°C. E2-BSA-FITC was detected by reverse phase HPLC using a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at 1.0 ml/min. The assay was run in triplicate. Representative spectra are shown for E2-BSA-FITC alone (A) and E2-BSA-FITC incubated with MC7 cells (B). Discussion The cellular effects elicited by estrogen [11,12,18-20,3] testosterone [3,21,22] and progesterone [23-25] covalently conjugated to membrane impermeable BSA have been attributed to non-genomic actions mediated by membrane associated hormone receptors. The use of these reagents for this purpose remains controversial for several reasons. A recent report demonstrated that E2-BSA does not bind to purified ER in competition assays with labeled E2 [14]. The studies were performed when E2-BSA or cold E2 were added concurrently with labeled E2. We obtained similar results under these conditions. However, pre-incubation of E2-BSA with purified ER results in significant competition with labeled E2. These conflicting results may be explained by differences in the rate of binding between E2 and E2-BSA. E2-BSA is a large, bulky molecule similar in size to the ER and is probably spherical in general structure as is the parent BSA molecule. The BSA protein conformation immediately adjacent to the covalently bound estrogen undoubtedly provides substantial steric hindrance to the proper presentation of conjugated E2 to the binding pocket of ER. The increased size of the E2-BSA molecule would also reduce the rate of its diffusion compared with the smaller E2. Correct orientation of E2 in the ER binding pocket is also impeded by the restraint on three-dimensional movement imposed by the six atom spacer used to connect BSA and E2. Lastly, the use of E2-BSA solutions that are formulated in terms of the molarity of total bound-E2 probably overestimates the amount of E2 available for binding. The rate of binding between E2 and ER can be expressed using the second order rate equation: rate = k [E2] [ER], where [E2] is the concentration of estradiol, [ER] is the concentration of the ER, and k the rate constant. Commercially available E2-BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E2-BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E2-BSA with respect to the concentration of estradiol alone. However the rate of E2-BSA binding is dependent upon the concentration of E2-BSA (rate = k [E2-BSA] [ER]). Even if every collision between E2-BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate. Taken together, these factors reduce the binding efficiency of E2-BSA to ER compared with free E2. However, once binding has occurred, the stability of the E2 molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E2-BSA results in successful binding, whereas immediate addition of E2-BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators [15] that demonstrate specific cell surface binding of E2-BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E2-BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E2-BSA and ER. Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than E2-BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells [26]. Membrane specific staining was prevented by treatment with antisense ERα mRNA or peptides that interfere with antibody binding. E2 conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E2 [27]. The membrane impermeable E2-BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ [15]. Moreover, ERα and ERβ interact directly with the membrane associated Src complex to trigger prostate cancer cell proliferation through the RAF-1/Erk-2 signal transduction pathway [5]. Lastly, we demonstrate that E2-BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E2 are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered. Conclusions The results presented here suggest that E2-BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E2 and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein. Materials and Methods Establishment of ER stable transfectants Full-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml). Preparation of E2-BSA free of E2 400 ul of E2-BSA (10-5 M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul. Binding of estradiol to purified estrogen receptor 3H-labeled E2 (NEN, specific activity 48 Ci/mmol, 10-8 M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E2 to ERα was competed by various concentrations of ultrafiltered E2-BSA or E2 (10-9 to 3.5 × 10-6 M in E2) added four hours prior to or concurrently with the addition of labeled E2. ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and 3H-E2 binding determined by liquid scintillation counting. E2-BSA-FITC binding to cell membranes of ER producing cells HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 10-8 M (in estrogen) E2-BSA conjugated to FITC (E2-BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E2-BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E2-BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E2, ICI-182,780, or E2-BSA (Sigma, 10-7 to 10-9 M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp). The stability of E2-BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E2-BSA-FITC (500 ul, 10-8 M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate. Estradiol binding studies Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 106 cells) incubated with several concentrations of 3H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis. Competing interests None declared. Authors' contributions DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays. ==== Refs Mermelstein PG Becker JB Surmeier DJ Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor J Neurosci 1996 16 595 604 8551343 Kelly MJ Moss RL Dudley CA Fawcett CP The specificity of the response of preoptic-septal area neurons to estrogen: 17 alpha-estradiol versus 17 beta-estradiol and the response of extrahypothalamic neurons Exp Brain Res 1977 30 43 52 563340 Armen TA Gay CV Simultaneous detection and functional response of testosterone and estradiol receptors in osteoblast plasma membranes J Cell Biochem 2000 79 620 7 10996853 10.1002/1097-4644(20001215)79:4<620::AID-JCB110>3.3.CO;2-8 Migliaccio A Di Domenico M Castoria G de Falco A Bontempo P Nola E Auricchio F Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells EMBO J 1996 15 1292 300 8635462 Migliaccio A Castoria G Di Domenico M de Falco A Bilancio A Lombardi M Barone MV Ametrano D Zannini MS Abbondanza C Steroid-induced androgen receptor-oestradiol receptor beta-Src complex triggers prostate cancer cell proliferation Embo J 2000 19 5406 17 11032808 10.1093/emboj/19.20.5406 Vargas R Delaney M Farhat MY Wolfe R Rego A Ramwell PW Effect of estradiol 17 beta on pressor responses of rat mesenteric bed to norepinephrine, K+, and U-46619 J Cardiovasc Pharmacol 1995 25 200 6 7752645 Farhat MY Abi-Younes S Dingaan B Vargas R Ramwell PW Estradiol increases cyclic adenosine monophosphate in rat pulmonary vascular smooth muscle cells by a nongenomic mechanism J Pharmacol Exp Ther 1996 276 652 7 8632333 Caulin-Glaser T Garcia-Cardena G Sarrel P Sessa WC Bender JR 17 beta-estradiol regulation of human endothelial cell basal nitric oxide release, independent of cytosolic Ca2+ mobilization Circ Res 1997 81 885 92 9351464 Nakhla AM Rosner W Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin Endocrinology 1996 137 4126 9 8828467 10.1210/en.137.10.4126 Pietras RJ Szego CM Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells Nature 1977 265 69 72 834244 Prevot V Croix D Rialas CM Poulain P Fricchione GL Stefano GB Beauvillain JC Estradiol coupling to endothelial nitric oxide stimulates gonadotropin-releasing hormone release from rat median eminence via a membrane receptor Endocrinology 1999 140 652 9 9927290 10.1210/en.140.2.652 Benten WP Lieberherr M Giese G Wunderlich F Estradiol binding to cell surface raises cytosolic free calcium in T cells FEBS Lett 1998 422 349 53 9498814 10.1016/S0014-5793(98)00039-8 De Goeij AF van Zeeland JK Beek CJ Bosman FT Steroid-bovine serum albumin conjugates: molecular characterization and their interaction with androgen and estrogen receptors J Steroid Biochem 1986 24 1017 31 3724140 10.1016/0022-4731(86)90355-9 Stevis PE Deecher DC Suhadolnik L Mallis LM Frail DE Differential effects of estradiol and estradiol-BSA conjugates Endocrinology 1999 140 5455 8 10537181 10.1210/en.140.11.5455 Razandi M Pedram A Greene GL Levin ER Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells Mol Endocrinol 1999 13 307 19 9973260 10.1210/me.13.2.307 Tassignon J Haeseleer F Borkowski A Natural antiestrogen receptor autoantibodies in man with estrogenic activity in mammary carcinoma cell culture: study of their mechanism of action; evidence for involvement of estrogen-like epitopes J Clin Endocrinol Metab 1997 82 3464 70 9329387 10.1210/jc.82.10.3464 Berthois Y Pourreau-Schneider N Gandilhon P Mittre H Tubiana N Martin PM Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems J Steroid Biochem 1986 25 963 72 3025520 10.1016/0022-4731(86)90330-4 Seo HS Leclercq G Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells J Steroid Biochem Mol Biol 2002 80 109 23 11867270 10.1016/S0960-0760(01)00175-3 Loomis AK Thomas P Effects of estrogens and xenoestrogens on androgen production by Atlantic croaker testes in vitro: evidence for a nongenomic action mediated by an estrogen membrane receptor Biol Reprod 2000 62 995 1004 10727269 Berthois Y Pourreau-Schneider N Gandilhon P Mittre H Tubiana N Martin PM Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems J Steroid Biochem 1986 25 963 72 3025520 10.1016/0022-4731(86)90330-4 Swenson KL Sladek CD Gonadal steroid modulation of vasopressin secretion in response to osmotic stimulation Endocrinology 1997 138 2089 97 9112409 10.1210/en.138.5.2089 Christian HC Rolls NJ Morris JF Nongenomic actions of testosterone on a subset of lactotrophs in the male rat pituitary Endocrinology 2000 141 3111 9 10965881 10.1210/en.141.9.3111 Beck CA Wolfe M Murphy LD Wiebe JP Acute, nongenomic actions of the neuroactive gonadal steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), on FSH release in perifused rat anterior pituitary cells Endocrine 1997 6 221 9 9368676 Contreras HR Llanos MN Detection of progesterone receptors in human spermatozoa and their correlation with morphological and functional properties Int J Androl 2001 24 246 52 11454077 10.1046/j.1365-2605.2001.00294.x Somanath PR Gandhi KK Expression of membrane associated non-genomic progesterone receptor(s) in caprine spermatozoa Anim Reprod Sci 2002 74 195 205 12417121 10.1016/S0378-4320(02)00168-9 Norfleet AM Thomas ML Gametchu B Watson CS Estrogen receptor-alpha detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry Endocrinology 1999 140 3805 14 10433242 10.1210/en.140.8.3805 Nadal A Ropero AB Laribi O Maillet M Fuentes E Soria B Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor alpha and estrogen receptor beta Proc Natl Acad Sci U S A 2000 97 11603 8 11027358 10.1073/pnas.97.21.11603

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==== Front BMC BiolBMC Biology1741-7007BioMed Central London 1741-7007-2-181531223310.1186/1741-7007-2-18Research ArticleBistability and hysteresis of the 'Secteur' differentiation are controlled by a two-gene locus in Nectria haematococca Graziani Stéphane [email protected] Philippe [email protected] Marie-Josée [email protected] Institut de Génétique et Microbiologie, UMR CNRS 8621, Bât 400 Université Paris-Sud, 15 rue George Clemenceau, 91405 Orsay Cedex, France2004 16 8 2004 2 18 18 5 5 2004 16 8 2004 Copyright © 2004 Graziani et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Bistability and hysteresis are increasingly recognized as major properties of regulatory networks governing numerous biological phenomena, such as differentiation and cell cycle progression. The full scope of the underlying molecular mechanisms leading to bistability and hysteresis remains elusive. Nectria haemaotcocca, a saprophytic or pathogenic fungus with sexual reproduction, exhibits a bistable morphological modification characterized by a reduced growth rate and an intense pigmentation. Bistability is triggered by the presence or absence of σ, a cytoplasmic determinant. This determinant spreads in an infectious manner in the hyphae of the growing margin, insuring hysteresis of the differentiation. Results Seven mutants specifically affected in the generation of σ were selected through two different screening strategies. The s1 and s2 mutations completely abolish the generation of σ and of its morphological expression, the Secteur. The remaining five mutations promote its constitutive generation, which determines an intense pigmentation but not growth alteration. The seven mutations map at the same locus, Ses (for 'Secteur-specific'). The s2 mutant was obtained by an insertional mutagenesis strategy, which permitted the cloning of the Ses locus. Sequence and transcription analysis reveals that Ses is composed of two closely linked genes, SesA, mutated in the s1 and s2 mutant strains, and SesB, mutated in the s* mutant strains. SesB shares sequence similarity with animal and fungal putative proteins, with potential esterase/lipase/thioesterase activity, whereas SesA is similar to proteins of unknown function present only in the filamentous fungi Fusarium graminearum and Podospora anserina. Conclusions The cloning of Ses provides evidence that a system encoded by two linked genes directs a bistable and hysteretic switch in a eukaryote. Atypical regulatory relations between the two proteins may account for the hysteresis of Secteur differentiation. ==== Body Background Although it has long been known that cellular memory, or epigenetic states, can be created by emergent properties of regulatory or metabolic networks (see Delbrück in the discussion of [1], and [2,3]), the full implications of this type of inheritance have only recently been understood. To date, pertinent studies focused mainly on phenomena related to chromatin structure and DNA methylation, RNAi and other post-transcriptional gene silencing processes, and prions. However, virtually any physiological process can adopt a bistable (or multistable) behavior, as defined by the ability to adopt two (or more) stable states rather than a range of intermediate states, provided that a positive feedback loop exists (or its related counterpart, the mutually inhibitory double negative feedback loop) within the system [1,3,4]. The bistability may sometimes be associated with hysteresis, i.e., the ability of the system to maintain, in a sustained manner, a particular state despite the fact that the stimulus initiating this state is no longer present or is below the level that initially activated the system [5,6]. In some cases, the hysteresis is sufficiently dominant to permit faithful transmission of the different states during mitosis and even meiosis, thus appearing as an epigenetic phenomenon [4,7]. In the light of these concepts, alternative inheritable regulatory states were designated (reviewed in [8]) based on previous descriptions of regulatory or metabolic networks, permitting the definition of new types of inheritance units, the 'toggle switch' and the 'positive feedback switch'. More recently, mathematical models defined the conditions in which a system endowed with positive autoregulation can present bistability [9,10]. However, most studies on bistable regulatory states are presently performed with a few well-known systems [7,8], including the lactose operon, the lambda lysogenic/lytic decision switch, and the Xenopus mos MAP kinase cascade. Other relevant models are necessary for the comprehension of these phenomena. Fungi are an excellent source of well-defined bistable and multistable processes associated with hysteresis (reviewed in [4,11]; see also [12]). In yeasts, such phenomena are quite common and are called phenotypic switches [11,13] or prions [14]. In filamentous fungi, bistable phenomena seem to be particularly prevalent, as one-third of the species show at least one example of these [4]. In most cases investigated in filamentous fungi, bistability results from the appearance and subsequent spread of cytoplasmic and infectious factors. Apart from the HET-s and the Crippled Growth determinants of Podospora anserina, their molecular nature and their roles remain unknown. HET-s is a prion [15] and the Crippled Growth determinant seems to result from a positive autoregulation of a signal transduction cascade [16]. The widespread recognition of these infectious factors in fungi is probably due to the ease with which they are detected. The syncytial structure of the mycelium facilitates the propagation of factors from cell to cell. Usually, the presence of the infectious element results in a modification of thallic characteristics, such as fertility or morphology, and is thus easily scored visually as sectors in which the mycelium displays different properties. In the filamentous ascomycete Nectria haematoccoca, two bistable phenomena have been described [17]. N. haematoccoca is the teleomorph of Fusarium solani, a fungus encountered throughout the world as both saprophyte and pathogen. N. haematoccoca produces naphthoquinones exhibiting biological activities. Production of naphthoquinones is regulated by genetic and environmental but also epigenetic factors. The thallus of this species, observed just after ascospore germination, is a dense, grayish aerial mycelium, called Normal. Two kinds of morphological modification (the Anneau and the Secteur) frequently and randomly appear in the growth margin of the thallus as small brown areas in which hyphal elongation is reduced and secretion of pigments is drastically increased (see Figure 1A for the development of the Secteur). Once initiated, the modifications spread and progressively invade the margin of the culture, with a speed that dictates their form (20 times the hyphal elongation speed for the Anneau and twice for the Secteur). The new growth forms can be perpetuated with large pieces of mycelium excised from the growing margin of affected mycelia. The resulting modified subcultures, called ZiS (from a Secteur) and ZiA (from an Anneau), grow slowly and are pigmented in brown. However, fragmentation of a modified culture permits the restoration of the Normal morphology in a variable proportion of subcultures [18], showing that ZiS and ZiA cultures are probably mosaics of infected and uninfected hyphae. Each modification can be specifically transmitted to Normal cultures by contamination experiments, as shown in Figure 1C for Secteurs. The tangential propagation of the new growth forms is thus due to the transmission through hyphal fusion (anastomoses) of specific infectious cytoplasmic determinants, called σ for the Secteur and α for the Anneau. Mutagenesis experiments of wild-type conidiospores demonstrated that both the Secteur and the Anneau are under the control of nuclear genes [19]. In these early mutageneses, several kinds of mutation were detected. Five mutations prevent the expression of both the Secteur and Anneau, either spontaneously or after inoculation. They differed from wild type in producing more aerial mycelium, lacking pigmentation, and suppressing the sexual stage through self-fertilization. They mapped to at least four loci. Since this type of mutation prevents the generation of both infectious factors, they are now designated nas mutants (for 'no Anneau or Secteur'). Several mutations that specifically prevent the formation of Anneaux were also selected [19]. The two different types of mutations recovered, a and a*, suggested that the Anneau is under the control of a unique but complex locus. All a and a* mutations map to the same locus, Ans (for 'Anneau-specific'). Mutants carrying the a mutations are unable to generate the α factor, since they never present Anneau morphology and cannot be infected by that factor. Mutants carrying the a* mutations never show Anneaux, spontaneously or after contamination, but display a red color. Surprisingly, when used as donor in contamination with the wild type as recipient, these mutants are able to trigger Anneaux in 100% of attempts, showing that these a* mutants carry the α factor constitutively. It is noteworthy that the phenotype of a* mutants suggests that the mere presence of this factor is sufficient to promote a red color but is insufficient to alter growth. To date, only one mutation that specifically prevents Secteur formation has been described [18,19]. This s*789 (formerly 789) mutant acts similarly to a* mutation in that it entails a red pigmentation (Figure 1B) and specifically prevents the expression of the Secteur, although it possesses the σ determinant throughout the thallus. This property is shown in Figure 1C, by the ability of small inocula harvested in the red s* thallus to induce Secteurs on growing recipient mycelia lacking the σ determinant. Because of the similar properties of Anneaux and Secteurs, we decided to screen for more mutations that specifically prevent Secteur formation, to determine if s mutations (i.e., a mutation that specifically prevents the generation of σ) could be obtained. To this end, we developed novel mutagenesis procedures permitting the recovery of mutations that specifically prevent Secteur development. Most were nas mutations. However, a few s* alleles and two s alleles (s1 and s2) were obtained. Genetic analysis suggested that the σ factor, like the α factor, is under the specific control of a single complex genetic locus. Molecular analysis of the s2 allele, obtained by insertional mutagenesis, permitted the cloning and characterization of the Ses locus. As expected from the genetic data, the locus is complex and encompasses two linked genes, one of which has similarity with putative esterase/lipase/thioesterase enzymes. These data provide evidence that two linked genes could generate a bistable differentiation. Results Selection and genetic characterization of mutants affected in the expression of the Secteur phenomenon To increase the number of mutants affected in Secteur expression, we decided to use a strategy based on the recovery of fast-growing sectors after mutagenesis of the slow-growing ZiS cultures (see Methods; Figure 2). We recovered 354 independent mutants (Table 1), with a large majority of nas mutants (97%). Eleven mutants were specifically affected in Secteur expression. Ten exhibited an s* phenotype identical to that of the s*789 strain. One mutant, s1, had a wild-type phenotype, except that it was unable to express the Secteur, spontaneously or after contamination tests (Figure 1D). Genetic analysis of the mutants defines a complex locus involved in Secteur expression The s1 mutant and five s* mutants were initially crossed with the I4mod reference strain. All behaved as single mutants, with about 50% wild type and 50% mutant type in the progeny (data not shown). In some progeny, the s* and s1 mutations were recombined with the I4mod markers. These strains were kept for further crosses. Crossing s1 with s*789 or crossing the new s* mutants with s1 and with s*789 yielded progeny that were mostly segregants, with the parental phenotypes in a ratio 1:1 and few segregants with the wild-type phenotype. Their frequency ranged from 0.1 to 0.6% of the progeny (Table 2), indicating that mutations affecting the Secteur were closely linked. In order to determine dominance/recessivity relationships of the mutated and wild-type alleles, forced heterokaryons were produced by pairing some mutants and wild-type strains marked with auxotrophic mutations (see Methods; Table 3). The s1/+, s*789/+, and s*84/+ prototrophic mycelia were grayish and able to differentiate Secteurs, as did the control heterokaryon, which carried only the auxotrophic markers. Thus, the wild-type allele was dominant over its mutated counterparts. Combinations of s* mutants with s1 gave rise to heterokaryotic wild-type mycelia, indicating that complementation occurred. In contrast, no complementation was observed in pairings involving two different s* alleles. The simplest interpretation of these results is that s1 and s* mutations could affect two different linked genes or two separate domains of a multifunctional protein encoded by a large gene. However, due to the low representation of heterokaryotic cells (about 10%) determined in previous studies [20], one must carefully interpret these data, deduced from the characteristics of the prototrophic heterokaryotic mycelium. Insertional mutagenesis identified the Ses locus Since the s1 mutant allele appears recessive to the wild type in balanced heterokaryons, we tried to clone the Ses locus by complementation using the sib selection strategy [21]. Although two different cosmid banks, XSG and XN (see Methods), were used, none of the tested transformants exhibited Secteurs. As an alternative strategy, we therefore used insertional mutagenesis, which can rapidly lead to the cloning of the relevant gene. Transformation with a plasmid that carries a hygromycin B resistance marker was performed to identify new mutants affected in Ses. However, because the contamination tests necessary for this screen were labor-intensive and time-consuming, we preferred the 61a1 strain as a recipient for transformation, since it expressed Secteurs very early, and at high frequency, and never expressed Anneaux [22]. As illustrated in Figure 3A, the majority of transformants grown for 15 days at 18°C differentiated Secteurs, so that few transformants had to be tested by contamination experiments for their Secteur expression. During the course of these experiments, we also screened transformants harboring a variation in colony morphology or pigmentation (Figure 3B). These phenotypes could be maintained from conidia isolation, showing that the transformation procedure was an effective approach in the creation of mutants. Ten transformants among the 5000 recovered failed to express the Secteur. Nine exhibited a nas phenotype. One was probably affected in the Ses locus, as it was unable to differentiate Secteurs, either spontaneously or after contamination tests. This transformant, called s2, had a wild-type morphology and behaved as did the s1 mutant previously isolated. The s2 transformant carries a DNA fragment integrated in the Ses locus In order to prove that s2 carries integration in the Ses locus, we crossed it with the wild-type (wt) and s1 strains. When crossed with wt, the 100 tested ascospores segregated 1:1 for ability:inability to differentiate Secteurs. As expected, hygromycin B resistance always cosegregated with the inability to differentiate Secteurs. When s2 was crossed with the s1 strain, only seven ascospores among 1657 were unable to express the Secteur (Table 2), indicating that s2 also mapped at the Ses locus. Overall, the data strongly suggested that s2 resulted from a single insertion at the Ses locus. To establish the integration pattern in s2, Southern blotting analysis was performed after digestion by ClaI, which did not cut the plasmid, and by BamHI, which cut once in the vector. The probe was the pAN7-1 vector used for insertional mutagenesis. As shown in Figure 4A, the number and intensity of hybridizing bands in ClaI digest reflected a complex integration event, with at least two integrated copies. Southern blot analysis of 10 s progeny from an s2 × wild-type cross did not show segregation of the two integration sites (data not shown), suggesting that the two integrated copies were linked. The size of the larger ClaI-fragment, LI, was compatible with an entire copy of the vector, whereas the smaller, SI, could be interpreted as a truncated copy. Further analyses with other restriction enzymes and probes (data not shown) established the restriction map of this region (Figure 4B). The sequence of a region flanking SI revealed that both LI and SI occurred at the same genomic site and were separated by a DNA fragment of unknown origin (Figure 4B). Molecular cloning of the Ses locus The restriction enzyme BamHI was used to recover the genomic sequence flanking LI (Flank1 in Figure 4B) along with part of the integrated pAN7-1 vector. A part of Flank1 was then used as a probe to screen the XN cosmid library. One hybridizing cosmid, XN31E6, was identified and used to transform the s*789, s*18, s*27, and s*4 mutants. Most of the transformants recovered the ability to express the Secteur, showing that the s* mutations are probably recessive. However, different phenotypes were observed (Figure 5). The 'mild S' exhibited the Secteur altered in its propagation and maintenance, and the 'S → s*' showed an s* phenotype beyond the Secteur. The mild S was interpreted as having a reduced amount of σ in the modified areas because of insufficient expression of the transgene, and the S → s* type was probably due to transgene sorting. Indeed, wild-type and s* nuclei could be recovered through the isolation of microconidia in such transformants, before expression of a Secteur, indicating that the S → s* thalli had a heterokaryotic structure. After the development of a Secteur, only the s* cells that were not affected in their growth in the presence of σ could grow, as previously observed for balanced heterokaryons [20]. Interestingly, we also observed several transformants displaying an s phenotype (Figure 5). These might have resulted from an abnormal integration inactivating the Ses locus. Molecular analysis of five hygromycin-resistant (HygR) transformants (four with a wild-type phenotype, and one with a mild-S phenotype) revealed two patterns of integration (Figure 6). The pattern exhibited by the four wild-type transformants was wild type, except for an additional band corresponding to the pMoCosX vector. These transformants were thus interpreted as having integrated one copy of XN31E6 at the resident Ses locus. The mild-S transformant pattern was more complex, showing additional bands and a difference in fragment stoichiometry, suggesting a complex integration. This is consistent with a reduced amount of σ hypothesized for mild-S transformants. It is noteworthy that despite several attempts, transformation experiments done with the s1 mutant as a recipient did not yield any transformants able to express the Secteur. This suggests that, contrary to the observations in balanced heterokaryons, s1 is dominant in partial diploids. Dominance of s1 was indeed established through transformation (see below). This dominance in diploids explains the failure to clone Ses by complementation. In order to further define the DNA fragment complementing the recessive s* mutation, cosmid XN31E6 was subcloned into pBluescript and the plasmids were assayed for complementation. This yielded psecX5, a plasmid carrying a 5.5-kb XhoI fragment sufficient to confer Secteur expression (Figure 7) with the same efficiency as did XN31E6. Gene organization of the Ses locus A 9-kb-long region surrounding the integration site in the s2 strain was sequenced (GenBank Accession no. AY572411). One interesting feature of the sequenced region concerned the presence of several inverted repeats ranging from 32 to 290 bp and organized in a symmetrical fashion (Figure 7). Inspection of the sequences revealed four open reading frames (ORFs) that were either larger than 200 amino acids or showed significant similarity with proteins in the databases (Figure 7). To begin with, we found an ORF encoding a putative large protein of unknown function that is also detected in the complete genome sequences available for the other filamentous ascomycetes (those of Neurospora crassa, Magnaporthe grisea, F. graminearum, Aspergillus nidulans, Aspergillus fumigatus, and P. anserina). This protein, NhHET-E-like, has a weak similarity (23% identity and 39% similarity over 455 amino acids) to the HET-E protein of P. anserina, which has been shown to be involved in heterokaryon incompatibility [23]. On the other side of the insertion site, a partial ORF of 165 amino acids carrying a PROSITE (PS00061), short-chain alcohol dehydrogenases/reductases family signature, was detected and designated ADHS. Two additional ORFs, SesA and SesB, were also identified. Functional analysis revealed that only these two ORFs were part of Ses, each having a different role. SesA is a 210-codon ORF (Figure 7) with no homologue in the genomes of N. crassa, M. grisea, A. nidulans, or A. fumigatus. However, it is significantly similar (30% identity and 42% similarity) to the N terminus of a large putative protein of P. anserina of unknown function but with 46% identity and 60% similarity with the Het-E protein in its C terminus (Figure 8A). It is also similar to two ORFs, of 214 and 201 codons, present in the genome of F. graminearum, with 41% identity and 54% similarity over 197 amino acids and 32% identity and 49% similarity over 199 amino acids, respectively (Figure 8A). SesA is interrupted by pAN7-1 in the s2 transformant, and sequence analysis of SesA in the s1 mutant showed a mutation at the end of the coding sequence, which changes a glycine into a glutamic acid (Figures 6, 7, 8A). SesA was expressed because a 480-bp-long RT-PCR product was obtained by using the 3' RACE (rapid amplification of cDNA ends) procedure. The sequence of this product revealed that a polyadenylation signal existed 141 bp downstream from the SesA stop codon. SesB, a 386-codon ORF (Figure 7), is located on the opposite strand from SesA. SesB shows significant similarity with several putative proteins from filamentous ascomycetes (at least three in A. fumigatus and A. nidulans, six in F. graminearum, two in N. crassa, and four in M. grisea and P. anserina) and animals (Caenorhabditis elegans, Drosophila melanogaster, or mammals). It does not show similarity with hemiascomycetous yeast ORFs (Saccharomyces cerevisiae and 12 other partially sequenced yeasts), basidiomycete ORFs (Coprinus cinereus, Ustilago maydis, and Phanerochaete chrysosporium), or Arabidopsis thaliana ORFs. All the proteins similar to SesB show a significant PROSITE (PS50187) esterase/lipase/thioesterase active site serine domain. The sequence of SesB in s1,s*789,s*4,s*27, and s*18 revealed that in the four s* mutant alleles, SesB contains a single point mutation (Figures 7 and 8B), but no mutation in the s1 mutant. In s*789, s*18, and s*4, the mutations are missense mutations, and in s*27, the mutation corresponds to a one-base addition resulting in a frameshift 120 bp upstream of the stop codon. None affected highly conserved residues of the esterase/lipase/thioesterase domain, suggesting that a functional protein was expressed in the mutants. The expression of SesB was also investigated with the 3'-RACE procedure. A 370-bp-long RT-PCR product was obtained, confirming that SesB is expressed. Sequencing showed that a polyadenylation signal is used 81 bp downstream from the SesB stop codon. Downstream of SesB is another potential short ORF (ORF-C), encoding a putative peptide of 157 amino acids with no homologue in the databanks. To show that ORF-C is not an exon of SesB, we cloned into pBluescript the 2.4-kb-long SmaI fragment carrying only SesB, i.e., without ORF-C or SesA. This yielded plasmid psecSm24 (Figure 7). Its transformation in four different s* mutants showed that the insert restored the expression of the Secteur. The molecular analysis of transformants showed that the integration was ectopic (data not shown), supporting the hypothesis that SesB alone is per se a functional gene involved in Secteur development. Since homologues of both SesA and SesB are present in the genomes of F. graminearum and P. anserina, we investigated whether the gene arrangement was conserved in these two species. In P. anserina, for which a single SesA homologue is found, the gene arrangement is not conserved, since SesA and SesB homologues are on two different contigs. In F. graminearum, the putative 201-amino-acid homologue of SesA that displays the lowest percentage of identity is not associated with any of the SesB homologues. On the contrary, the 214-amino-acid homologue of SesA that displays the highest identity is associated with a SesB homologue in the same order (Figure 8C). This homologue is highly conserved between N. haematococca and F. graminearum (79% identity and 85% similarity over 387 amino acids) whereas the other F. graminearum homologues are less conserved (40% identity and 63% similarity over 273 amino acids for the next-best homologue). In F. graminearum, the inverted repeats 1, 2, and 3 are absent but the inverted repeat 4 is present. Overall, these data suggest an evolutionary conservation of Ses between N. haematococca and F. graminearum, both of which are in the genus Fusarium, but with a lack of conservation of the gene organization in more distant species, including the loss of SesA. There are no data concerning the potential development of Secteur in the F. graminearum strain used by the Fungal Genome Initiative for sequencing. s1 is dominant in diploids To investigate the dominance of s1, we co-transformed the wild-type strain with pSec3-s1 and pBC-Hygro. pSec3-s1 was obtained by cloning into 'pGEM-T easy' (Promega, Charbonnières, France), a PCR amplification product constructed with oligonucleotides im317 and ip2393c (see Figure 7) using the s1 mutant genomic DNA as template. Thus, pSec3-s1 contained the entire Ses locus present in the s1 mutant strain. Among 17 tested HygR transformants, seven could not differentiate Secteurs. StuI-restricted DNA from five of these transformants was analyzed by Southern blotting. Two integration patterns were observed (Figure 9). For three transformants, the expected wild-type StuI fragment was absent, suggesting that integration altered the Ses locus, thus accounting for the lack of Secteur formation. For the two other transformants, the wild-type StuI fragment was present, as was an additional band larger than 15 kb. As this band hybridized with psec3-s1 and the hph hygromycin resistance marker (data not shown), these transformants could be interpreted as bearing a co-integration of pBC-Hygro and pSecs1 at an ectopic position. Sequencing of the Ses locus in these two transformants that do not display Secteurs confirmed the presence of both alleles, i.e., the wild-type allele at the resident locus and the mutated allele s1 at an ectopic position. This clearly demonstrated the dominance of the s1 mutation, in contrast to the s* mutations, which are recessive. Discussion Although the Secteur and Anneau phenomena were described about 30 years ago [17], the molecular mechanisms able to generate these fascinating bistable differentiations are still mysterious. Early studies suggested peculiar epigenetic mechanisms [24]. In this study, we have initiated the molecular characterization of Ses, the primary locus controlling the development of Secteurs. The impact of mutagenesis procedures The experimental design, based on the differences in the growth rate between modified and normal cells, was very efficient in finding mutations that block the propagation of Secteurs after the action of both UV and NG (N-methyl-N'-nitro-N-nitrosoguanidine). A vast collection of mutants affected in the Secteur expression was easily obtained. One mutant with an s phenotype was isolated, along with 11 s* mutants. Apparently, NG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis was more efficient than UV mutagenesis in recovering these specific mutants (9/105 and 3/250, respectively). Genetic analysis showed that the structure of the Ses locus mirrors the structure of the Ans locus, indicating that α and σ could be generated through similar mechanisms, in agreement with the fact that both factors require the same set of nas genes for propagation. Relationships between s1 and wt alleles: recessivity in heterokaryons versus dominance in partial diploids Dominance/recessivity tests using balanced heterokaryons showed that s1/wt heterokaryons were able to differentiate Secteurs as wild type. The recessivity of s1, deduced from the heterokaryon tests was the initial point of our cloning strategy, i.e., the use of cosmid libraries from wild type to restore the ability to express the Secteur in an s1 recipient strain. The screening of two representative cosmid libraries failed, and we examined various hypotheses to explain this failure, especially the possibility of the dominance of s1. In order to easily clone the Ses locus, we used an insertional mutagenesis strategy, a powerful method for gene isolation in filamentous fungi [25]. With the pAN7-1 vector, 5000 HygR transformants were screened for defects in mycelium pigmentation, colony morphology, and the expression of the Secteur and around 4% of mutant phenotypes were detected. This frequency is similar to the 0.4 to 1.4% reported for loss of pathogenicity in different fungal species [25]. Among them, 9 nas mutants and 1 s mutant were obtained. Given the proportion observed for classical mutagenesis (12 s* or s mutants among 355 nas), the recovery of the s2 mutant was an unexpected outcome. Although we may have been lucky, the recovery of this transformant may reflect a preference for plasmid integration in the region of the Ses locus, which is compatible with the high recombination frequency observed at Ses. Cloning of Ses permitted the construction by transformation of a partial diploid containing both the wild-type and s1 mutant alleles. The fact that this partial diploid has a s phenotype indicates that s1 allele is indeed dominant over the wild-type allele in partial diploids. Similar contradictions have been observed in A. nidulans by comparing diploids and heterokaryons for complementation between mutations in the regulatory and the cognate structural genes. These data were interpreted as being due to a limitation of regulatory gene products to the nucleus or by a stringent dose effect when combined with a nonrandom distribution of nuclei in heterokaryons [26,27]. In our study, a dose effect is also probable. In N. haematococca, as reported earlier [20], only 1–10% of hyphal fragments containing 3–4 cells are heterokaryotic, the others corresponding to both homokaryons in similar proportions. In addition, phase-contrast microscopic observation of the prototrophic mycelia revealed the presence of rows of uninucleate cells disrupting the heterokaryotic association. This structure does not prevent the stability of balanced heterokaryons with regard to metabolic requirements (hence the complementation of the auxotrophic mutations), but might severely affect the dosage of the SesA products expressed from the wild-type and s1 nuclei in different portions of the thallus. Because s1 is dominant and corresponds to a missense mutation, it is likely that the product expressed from SesA in s1 is a dominant negative. If its concentration is insufficient, especially in hyphae homokaryotic for wild-type SesA nuclei, the propagation of σ and Secteur formation is favored. In contrast, in diploids an even ratio of the two products is always achieved, preventing propagation of σ and Secteur formation. Integrated model of functional elements at the Ses locus The molecular characterization of Ses shows that it is composed of two linked and expressed genes, SesA and SesB, both of which are necessary for Secteur expression. Each has a different role. The s1 and s2 mutants map to SesA. Because s2 is an integration of a large segment of DNA at the beginning of SesA, the phenotype of the s2 mutant probably results from a complete loss of function of SesA. The s1 mutant carries a dominant missense mutation and therefore synthesizes the SesA mRNA, and probably a dominant negative protein. The SesAp protein has no evident homologue in the databanks yielding a clue to its function. As s2 does not produce the σ determinant, cannot be contaminated by mycelia carrying σ, and never differentiates the Secteur morphology, SesA is probably necessary for the production and transmission of σ. All s* mutations map to SesB and determine a red-pigmented phenotype associated with the generation of the σ determinant throughout the thallus, but without any growth alteration. It is noteworthy that all the s* mutations are probably not simply preventing the production of a functional mRNA, but are also not preventing the production of an active protein, as they map outside the evolutionarily conserved region of the protein. The function of SesB and its homologue remains unknown in fungi; however the inactivation by RNA interference of the most similar gene in C. elegans is lethal during embryogenesis, suggesting that the protein produced by SesB could be essential (accession NP_510177; [28]). Based on the analysis of the mutant phenotype triggered by s* mutation, the wild-type SesB product is involved in the repression of pigmentation and σ generation. To date, this situation has not been described for any of the previously reported systems responsible for bistable or multistable switches in fungi. Previous systems implicated prions [23,29], chromatin silencing [12], hysteresis in a MAP kinase cascade [16], and possible membrane inheritance [30,31]. At present, it is premature to propose a molecular model to explain the Secteur, because SesA and SesB display weak similarity only with proteins of unknown function. However, from our data we can infer that SesA most likely encodes a regulatory factor for SesB and that the regulation probably takes place at the protein level and probably not at the RNA level, since s1 and all s* mutation are point mutations that are not likely to affect RNA transcription and stability. Two formal models could explain how the regulation might work. Firstly, the product of SesA (SesAp) could exist in two states: one, AN, characterizing the Normal morphology and the other, AS, determining the Secteur morphology (Figure 10A). The transition AN to AS may be initiated by a rare event in cells of the growing margin of the thallus. Once formed, the AS state becomes predominant by directing other AN molecules to adopt the same state. This positive feedback loop allows AS propagation from hyphae to hyphae through anastomoses. AS alone would induce the hyperproduction of pigments (hence the absence of both σ and pigmentation in the s mutants, as they are mutated in SesA). In addition, the product of SesB (SesBp) can adopt the normal BN state, which can be transformed into a BS state in the presence of AS. BS would be responsible for the growth alterations observed in the Secteur and would follow the propagation of AS (hence the lack of growth impairment presented in the s* mutants, as they are mutated in SesB). In the second model, SesAp may negatively regulate SesBp but also be negatively regulated by SesBp (Figure 10B). In the normal mycelium, SesBp, which probably has a catalytic activity, would be active in promoting healthy growth. The appearance in some cells from the growing margin of a sufficient amount of SesAp would displace the equilibrium, resulting in the inhibition of SesBp. In order for this to happen, the equilibrium constants must be in favor of SesAp. This would result in growth impairment (because SesBp would not be present) and in a red pigmentation (because SesAp would be present). In the s mutants, SesAp would never be present, thus making the presence of SesBp constitutive (hence the absence of Secteurs and the healthy growth). In the s* mutant, the interactions between the two proteins would be abolished, permitting the activity of both SesAp and SesBp (hence the presence of a constitutive σ, the red pigment, and healthy growth). This model could explain the dynamic equilibrium found in the ZiS area, as well as the dose effect for SesAp, explaining the difference of heterokaryons and partial diploids. Conclusions Secteur, one of the two bistable and hysteretic differentiations exhibited by the filamentous fungus N. haematococca, is controlled by a complex, two-gene locus. Each gene has a distinct role in defining bistability, and atypical regulatory relations between the two proteins coded by the two genes may account for the hysteresis of Secteur differentiation. The data exemplify the diversity of mechanisms that generate differentiation in fungi, and, more generally, in eukaryotes. Methods Fungal strains and growth conditions The homothallic N. haematococca strain wt (Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) was used as the standard wild-type strain in this study. The origin and relevant characteristics of all strains used in this study are listed in Table 4. The a1s1I4mod and 61a1 strains used as recipients in transformation experiments were constructed by crossing the s1I4mod strain with a1 mutant and the 61 mutant with a1I4mod strain, respectively (unpublished data). The marker segregation was recorded after three weeks. This included (1) mycelium and perithecium pigmentation, (2) fertility, and (3) the ability to express the Secteur and the Anneau as tested by transferring the progeny onto potato dextrose agar (PDA) and by inoculating 3-day cultures with s*789 (for Secteurs) and a*58 (for Anneaux). General culture conditions and manipulations were as described by Daboussi-Bareyre [20]. Strains were purified through microconidia and maintained on PDA at 26°C. Long-term stocks were stored as plugs under mineral oil at 12°C. The medium selective for transgenes was PH8, i.e., PDA containing 8 μg/ ml of hygromycin B from Sigma-Aldrich (Saint Quentin Fallavier, France). Mating and ascospore recovery The general methods for crossing the homothallic N. haematococca have been described elsewhere [19,32]. Since the strains used in this study were homothallic, detection of hybrid perithecia in crosses were performed using as a partner in crosses the double mutant strain I4mod. This strain developed white, self-sterile perithecia whereas wild-type and fertile mutants developed self-fertile, red perithecia [32]. Thus, all the white, fertile perithecia, which appeared on the crossing plates, were hybrid. The progeny of crosses was analyzed in accordance with the following procedure: two to nine white, fertile perithecia from each cross were freed from mycelium and conidia. Each was opened to spread the ascospores on one Petri dish containing water plus 3% agar. To control viability, 35 ascospores from each perithecium were transferred to PDA and incubated for 4 days at 26°C, while the water-agar stock dishes were kept at 2°C to prevent ascospores from germinating. Then, the water-agar dishes containing many viable ascospores (usually those with more than 70% of germination were selected) were returned to 26°C to allow germination overnight. A sample of about 400 germinating ascospores from each dish was transferred to PDA. The phenotypes were recorded after a week at 26°C in the dark, except for the perithecium color, which was evaluated after an additional period of two weeks. Heterokaryon test Heterokaryons were constructed as described in [20]. Initially, strains s1, s*84, and s*18 were crossed with the two strains carrying either of the markers, arg154 and lys255, that promote auxotrophy for arginine and lysine, respectively. Auxotrophic marked strains, which were recovered in the progeny, were paired on a cellophane membrane in the combinations appropriate to restore prototrophy. After 48 hours of growth on complete medium, the membrane was transferred to minimal medium. Widely inclusive inocula from prototrophic mycelia observed at the junction of the two partners were transferred to minimal medium and examined for their pigmentation and their ability to express the two modifications, either spontaneously or after inoculation. Standard mutagenesis Plugs taken from the growing margin of a culture modified by the Secteur were inoculated on cellophane discs on PDA and grown for 4 days at 26°C. The modified slow-growing cultures (ZiS) were submitted to mutagenesis by UV or NG. For UV mutagenesis, cultures were exposed at 450 J/m2, with a UV light source (254 nm), and then kept in the dark for 72 hours. For NG mutagenesis, 0.1 ml of a 200γ/ml solution of NG was deposited under the cellophane disc. After 1 hour, the treated cultures were exposed to light for NG degradation. Fast-growing sectors were clearly visible a week after mutagenesis. For the estimation of the number of sectors recovered from each treated thallus, only sectors harboring different phenotypes were considered. A plug of these independent sectors was inoculated on PDA and the resulting cultures were examined for pigmentation and ability to express the Secteur and/or the Anneau at 26°C. The majority of the recovered mutants corresponded to nas mutants, defined by (1) the inability to differentiate both Anneaux and Secteurs, (2) an exuberant white mycelium, and (3) the inability to differentiate perithecia. Some of these mutants could express the modifications at other temperatures, as was previously observed for mutants 727 and 100 [33,34]. s* mutants were recovered as red, fast-growing sectors. They displayed the same properties as the s*789 mutant described previously [19], i.e., red thallus and inability to express the Secteur spontaneously or after inoculation. A unique s mutant was selected as a wild-type-growing sector. This mutant displayed a wild-type phenotype, except for the inability to express the Secteur spontaneously or after inoculation. Both s* and s mutants could differentiate Anneaux. Insertional mutagenesis Plasmids pAN7-1 (GenBank accession number Z32698) or pBC-Hygro [35], which had no sequence similarity with the N. haematococca genome, were used to transform the double mutant strain 61a1. Both vectors carried a hygromycin resistance gene. Transformation occurred at a frequency of about 10 transformants/μg plasmid. Southern analysis on 10 independent hygromycin-resistant transformants (HygR) indicated that the transforming DNA inserted generally at one or two genomic sites, in some cases in a tandem fashion (data not shown). This pattern of integration was suitable for recovery of tagged genes. To screen for mutant phenotypes, 5000 HygR independent transformants were transferred to PH8 (12 per Petri dish) and grown for 15 days at 18°C, a temperature that increased the frequency of spontaneous Secteur formation. Thalli with mutant phenotypes were frequently recovered (Figure 3). These most likely resulted from the plasmid integrations, since a sample of regenerating protoplasts treated in the same way but without the transforming DNA did not show any phenotypic variation. As control, a sample of transformants with an altered morphology was purified through single conidial isolation. Data showed that the mutant phenotype was stable. Potential candidates affected in the expression of the Secteur were recovered as thalli that did not form spontaneous Secteurs. Inoculation experiments were used to confirm the mutant phenotypes. Transformation procedure Protoplasts were prepared as described in [36], with the following modifications. Petri dishes containing 25 ml of a solid PDA medium covered with a cellophane disk were inoculated with 5×106 spores and incubated for 20 hours at 26°C. Mycelia were collected (0.2 g/10 ml) in a lysis buffer (1.2 M MgSO4, 10 mM sodium phosphate, pH 5.8, 10 mg/ml Glucanex (Novozymes, Bagsvaerd, Denmark), 10 mg/ml mutanase (Interspex Products, San Mateo, CA, USA), and incubated for 2–3 hours at 26°C with gentle agitation. Protoplasts were separated from cell debris by addition of 5 ml of ST buffer (0.8 M Sorbitol, 100 mM Tris-HCl pH = 7.5) and centrifugation at 2000 g for 10 min. The protoplasts collected at the interface of the two solutions were harvested by centrifugation at 750 g for 5 min, washed twice in ice-cold buffers, once in ST and then in STC (0.8 M Sorbitol, 50 mM CaCl2, 100 mM Tris-HCl pH = 7.5; [36]). They were resuspended in 4/5 STC, 1/5 50% PTC buffer (50% PEG 3350 [Sigma-Aldrich], 100 mM CaCl2, 100 mM Tris-HCl, pH 7.5) at a final concentration of 5–10 × 107 protoplasts/ml and kept on ice. For transformation, 5 μg or 10 μg of DNA suspended in a maximum of 50 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were mixed gently with 100 μl protoplasts and placed on ice. After 30 min, 900 μl of PTCS buffer (50% PTC containing 0.8 M sorbitol) was added and incubation continued at room temperature for 30 min. Aliquots of the protoplasts were mixed with 2.5 ml molten (37°C) top PDAS (PDA with 0.8 M sucrose and 0.3% w/v agar), and overlaid onto 30 ml of selective medium (PDAS containing 8 μg/ ml of hygromycin B. Plates were incubated at 26°C and colonies appeared within 2–4 days. Numerous small colonies stopped growing after 2–3 days. These were interpreted as abortive transformants. Only the colonies that grew after this delay were considered resistant to hygromycin. When co-transformation experiments were performed, 5 μg of the transforming plasmid was mixed with 5 μg of pBC-Hygro. DNA manipulation All the nucleic acid manipulations were performed using standard methods [37]. For Southern blot analysis, genomic DNA was extracted as described in [38]. The cosmid libraries were made in the pMoCosX vector as recommended by Orbach [39]. The library XSG was obtained with XhoI partially digested wt genomic DNA. Determination of gene representation in the XSG library was performed by estimating the number of clones containing conserved genes (NhNia encoding the nitrate reductase, NhHsp70 and NhHsp90 encoding molecular chaperones, NhTUB1 encoding β-tubulin, NhTEF1 encoding the translation initiation factor eEF1A) by PCR, Southern blot hybridization, or direct cloning using a complementation screen. Each tested gene was represented by 2 to 5 independent clones, indicating that the library contained at least four N. haematococca genome equivalents. Library XN was made with SalI partially digested wt genomic DNA following the same protocol as for the XSG library. The sequence of the mutant alleles was performed on two independent PCR amplification products, obtained with oligonucleotides im317 (5'-TGATCAACCTCCACGCACAT-3') and ip2393c (5'-AAGGAGATATCGCGCAGGCT-3'). Identification of the 3' end of the SesA and SesB genes was done by 3'-RACE using the 5'/3' RACE kit (Roche Diagnostics, Meylan, France) on polyA+ mRNA extracted from 2-day-old mycelium, using for SesA oligonucleotide ip238 (5'-AAGAGGCCGTGAGACAAGAG-3'), and for SesB ip974c (5'-CCAAGACGACCGATAGAAGA-3'). GenBank accession The genBank accession no. for cosmid XN31E6 sequences is AY572411. Authors' contributions SG performed all the experimental work and contributed to the interpretation of the data. PS and MJD performed part of the phylogenetic analysis, contributed to the interpretation of the data, and supervised the work. The writing of the manuscript was done in teamwork. All authors read and approved the final manuscript. Acknowledgements We thank Emilie Robillard for expert technical assistance. This work was supported by ACC on ESST and Prions ref 2000-37. Stéphane Graziani was a PhD student, recipient of a 'Bourse de Docteur Ingénieur du Centre National de la Recherche Scientifique' training grant. Philippe Silar is a professor at the University of Paris 7, Denis Diderot. The work was done in compliance with the current laws governing genetic experimentations in France. Figures and Tables Figure 1 The Secteur and its transmission through contamination tests in Nectria haematococca. (A) Wild-type strain exhibiting three spontaneous Secteurs (1, 2, 3). (B) s*789 mutant. (C) Contamination tests, using plugs from a Secteur or from s*789, on wild type (wt), result in one Secteur at each infection point. (D)Contamination tests on s1 mutant did not result in the induction of any Secteur at the infection points. Figure 2 Selection of Nectria haematococca mutants affected in the expression of the Secteur. (A) Compares the morphology of a Normal growing culture (N) and of a ZiS culture (modified culture obtained by excision from a Secteur). (B) A 4-day ZiS culture submitted to UV mutagenesis. Note the emergence of four fast-growing sectors, which correspond to nas mutants. Figure 3 Phenotypic characterization of transformants. (A) Detection of a nas mutant phenotype among 12 transformants. Arrows indicate some Secteurs. (B) Summary of the different mutants recovered among the 5000 transformants: 152 were altered in pigmentation (99 reddish, 16 white/beige/yellow, 37 greenish), 45 were altered in colony morphology (12 irregular, 12 compact, 19 colonial, 2 fluffy), and 10 were affected in the expression of the Secteur (9 nas and 1 s). Figure 4 Analysis of pAN7-1 integration pattern in s2.(A) Southern blot analysis of total genomic DNA of s2 digested with ClaI, which does not cut the plasmid (lane 1), and BamHI, which cuts once in pAN7-1 (lane 2), and probed with the pAN7-1 vector. Sizes are indicated in kilobases (kb). (B) Restriction map of the pAN7-1 integration point in s2. Wild-type genomic DNA is shown as a thick line, pAN7-1 sequences are represented by the open bar, and the dotted line stands for unknown sequences. The fragment LI is inserted at the bacterial replication origin (ORI). SI is truncated and carries a part of hph and of tTrpC. Figure 5 Types of transformants recovered in transformation experiments using s* mutants with XN31E6 and psecX5. As the respective proportion of each type of transformant was similar with both vectors, the proportion observed is the combined percentage computed with 60 tested transformants. S, transformants differentiating Secteurs as wild type; mild S, transformants differentiating a Secteur that propagates more slowly than wild type and that frequently reverts to a normal morphology. S → s*, transformants differentiating the Secteurs but showing an s* phenotype beyond the modified area; s, transformants unable to differentiate Secteurs. Figure 6 Southern blot analysis of XN31E6 transformants. XN31E6 cosmid DNA along with total genomic DNAs of wild type (wt) s2, and five transformants obtained by transforming s*789 with XN31E6 were digested by XhoI and hybridized with an XN31E6 probe. Wild type, Tr1, Tr2, Tr3, and Tr4 differentiate wild-type Secteurs (S), Tr5 has a mild S phenotype, and s2 is not able to differentiate Secteur(s). The 7-kb band corresponding to pMoCosX is marked by dark connecting lines. The arrow points to the 5.3-kb fragment, which shifts in s2. Figure 7 Schematic representation of the Ses locus of Nectria haematococca. (A) Open reading frames larger than 200 aa or showing sequence similarity to other genes are represented by gray arrows. Black arrows represent sequences in inverted repeated orientation: repeat 1 is 290 bp long; repeat 2 is 79 bp long; repeat 3 is 32 bp long; and repeat 4 is 93 bp long. Thick lines: subclones able to restore the Secteur modification in s* mutants. (B) Detailed map of the region between oligonucleotides im317 and ip2393c. Vertical bold lines: position of the indicated mutations. Insertion: integration point of pAN7-1 in s2. Black lozenge: polyadenylation site. Figure 8 SesA and SesB conservation during evolution. (A) Alignment of SesAp with its homologues in Fusarium graminearum (F. gra 214 and F. gra 201) and Podospora anserina (P. ans). The amino acid change in s1 is boxed. (B) Alignment of SesBp with representative homologues from F. graminearum (Fg1 and Fg2; the sequence was obtained from the Fungal Genome Initiative, ), P. anserina (Pa; ), Homo sapiens (Hs; GENBANK: BC001705), Drosophila melanogaster (Dm; GENBANK: AAF5369), and Caenorhabditis elegans (Ce; PIR: T21079). Arrowheads indicate the amino acid changed in s*4, s*789, and s*18; arrow indicates the position of the frameshift in s*27. (C) Conservation of Ses organization in F. graminearum. SesA corresponds to F. gra 214 of (A) and SesB corresponds to Fg1 of (B). Percentages of identity with Nectriahaematococca SesAp and SesBp are 41% and 79%, respectively. In (A) and (B), conserved amino acids are shaded. Color reflects the degree of conservation. Figure 9 Southern blot analysis of StuI-digested DNAs of wild-type Nectria haematococca and five transformants. Total genomic DNAs of wild-type (wt) Nectria haematococca and five s transformants (Tr1-Tr5) that were obtained after co-transformation of wt with psec3-s1 plasmid and pBC-Hygro vector, and probed with psec3-s1. All the transformants share a large band (>15 kb), absent from wild type. Tr3 and Tr5 contain both the wild type and the s1 allele. The faint bands noted * correspond to repeated sequences homologous to those present at the Ses locus. Figure 10 Models integrating functional elements of Secteur expression in Nectria haematococca. (A) First model: In the wild-type strain, the product of the SesA and SesB genes can exist in a normal state (AN and BN) and in a modified state (AS and BS). AS determines overproduction of pigments and BS is responsible for growth alteration. (B) Second model: The product of SesA, SesAp, is responsible for pigmentation. The product of SesB, SesBp, has a catalytic activity necessary for the normal growth. These two products negatively regulate each other, and the equilibrium constants favor SesAp. In juvenile thalli, only SesBp is produced, but SesAp can be randomly produced, switching to a new state with a majority of SesAp. Table 1 Mutagenesis efficiency and spectrum of mutants Mutagena Total number of irradiated thalli Total number of fast-growing sectors Types of mutantsb nas s* s UV1 12 15 15 - - UV2 26 53 53 (13) - - UV3 21 40 39 (6) 1 (s*11) - UV4 28 50 49 (16) 1 (s*27) - UV5 44 75 74 1 (s*4) - UV6 21 17 17 - - NG1 28 25 22 2 1(s1) NG2 22 38 37 1 (s*18) - NG3 26 42 37 5 - Total 228 354 343 11 1 1.5 per dish 97 3 aUV, 254 nm UV irradiation; NG, N-methyl-N'-nitro-N-nitrosoguanidine. bnas mutants differentiate neither Anneaux nor Secteurs at 26°C but some may do so at other temperatures. Their number are indicated in brackets; s* mutants differentiate Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; s, cultures differentiate Anneaux but not Secteurs, and never contain the σ determinant. Table 2 Results of crosses involving mutants of the Secteur Crosses Progency analyzed Wild-type progeny No. % s × s*27 3256 5 0.15 s1 × s*18 3488 7 0.20 s1 × s*789 3824 9 0.25 s1 × s*4 2217 11 0.50 s1 × s*84 1977 10 0.65 s1 × s*11 1051 7 0.65 s*789 × s*84 2944 3 0.1 s*789 × s*4 1095 1 0.1 s*789 × s*11 1808 2 0.1 s*789 × s*27 2346 1 0.05 s*789 × s*18 2139 6 0.3 s1 × s2 1657 7 0.40 Table 3 Phenotypes of heterokaryotic mycelia from various pairings Strains paired Prototrophic myceliuma Conclusion arg154 (+) / lys255 (+) Gray, AS arg131(s*789)/lys255(s*789) Red, As* arg154(s*84)/lys255(s*84) Red, As* arg154(s1) / lys255(s1) Gray, As arg154(s1) / lys255(+) Gray, AS s1 recessive arg154(s*789) / lys255(+) Gray, AS s*789 recessive arg154(s*84) / lys255(+) Gray, AS s*84 recessive arg154(s1) / lys255(s*789) Gray, AS Complementation arg154(s1) / lys255(s*84) Gray, AS Complementation arg154(s1) / lys255(s*18) Gray, AS Complementation arg131(s*789) / lys 255(s*84) Red, As* No complementation arg154(s*18) / lys255(s*84) Red, As* No complementation aAS differentiates both Anneaux and Secteurs and synthesize α and σ only in the respective modified areas. As* differentiates Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; As differentiates Anneaux but not Secteurs and never contain the σ determinant. Table 4 Nectria haematococca strains used Characteristicsb Strain Origina Mycelium Fertility Differentiation Source Wild type CBS225/58 Gray FFF AS s*789 NG Red F As* Daboussi-Bareyre et al. 1979 s*27 UV Red F As* This study s*4 UV Red F As* This study s*11 UV Red F As* This study s*18 UV Red F As* This study s*84 sp Red F As* Parisot et al. 1981 s1 NG Gray FFF As This study s2 IM Gray FF As This study 61 UV Gray F AS with a high frequency of Secteur Daboussi 1985 a1 Sp Gray FFF aS Daboussi-Bareyre et al. 1979 a*58 NG Red F a*S Daboussi-Bareyre et al. 1979 arg154 NG Beige, Smooth arginine-auxotroph F AS Daboussi-Bareyre 1980 lys255 NG Beige, Smooth lysine-auxotroph F AS Daboussi-Bareyre 1980 I4mod cross Gray St, white and empty perithecia AS Babai-Ahary et al. 1982 aCB: Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands. sp, spontaneous mutation; NG, UV and IM recovered after N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiadiation (254 nm) or insertional mutagenesis, respectively. cross: constructed by crossing. bMain phenotypic characteristics. FFF, very fertile; F, fertile; St, sterile. AS differentiates both Anneaux and Secteurs and synthesizes α and σ only in the respective modified areas. As* differentiates Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; As differentiates Anneaux but not Secteurs and never contain the σ determinant; a*S differentiates Secteurs but not Anneaux, though they have the α determinant throughout the mycelium; aS differentiate Secteurs but not the Anneaux and never contain the α determinant. ==== Refs Sonneborn TM Beale SH Influence des gènes, des plasmagènes et du milieu dans le déterminisme des caractères antigéniques chez Paramecium aurelia (variété 4) Unités biologiques douées de continuité génétique 1948 Paris – CNRS 33 34 Nanney DL Epigenetic control systems Proc Natl Acad Sci U S A 1958 44 712 717 Beisson J Non-nucleic acid inheritance and epigenetic phenomena Cell Biology 1977 1 375 421 Silar P Daboussi MJ Non-conventional infectious elements in filamentous fungi Trends Genet 1999 15 141 145 10203823 10.1016/S0168-9525(99)01698-4 Sha W Moore J Chen K Lassaletta AD Yi CS Tyson JJ Sible JC Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts Proc Natl Acad Sci U S A 2002 100 975 980 12509509 10.1073/pnas.0235349100 Bagowski CP Ferrell JE Jr Bistability in the JNK cascade Curr Biol 2001 11 1176 1182 11516948 10.1016/S0960-9822(01)00330-X Ferrell JE Jr Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability Curr Opin Cell Biol 2002 14 140 148 11891111 10.1016/S0955-0674(02)00314-9 Judd EM Laub MT McAdams HH Toggles and oscillators: new genetic circuit designs Bioessays 2000 22 507 509 10842304 10.1002/(SICI)1521-1878(200006)22:6<507::AID-BIES3>3.0.CO;2-0 Ozbudak EM Thattai M Lim HN Shraiman BI Van Oudenaarden A Multistability in the lactose utilization network of Escherichia coli Nature 2004 427 737 740 14973486 10.1038/nature02298 Angeli D Ferrell JE JrSontag ED Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems Proc Natl Acad Sci U S A 2004 101 1822 1827 14766974 10.1073/pnas.0308265100 Malagnac F Silar P Non-Mendelian determinants of morphology in fungi Curr Opin Microbiol 2003 6 641 645 14662362 10.1016/j.mib.2003.10.003 Halme A Bumgarner S Styles C Fink GR Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast Cell 2004 116 405 415 15016375 10.1016/S0092-8674(04)00118-7 Soll DR Mating-type locus homozygosis, phenotypic switching and mating: a unique sequence of dependencies in Candida albicans Bioessays 2004 26 10 20 14696036 10.1002/bies.10379 Roberts BT Wickner RB Heritable activity: a prion that propagates by covalent autoactivation Genes Dev 2003 17 2083 2087 12923060 10.1101/gad.1115803 Maddelein ML Dos Reis S Duvezin-Caubet S Coulary-Salin B Saupe SJ Amyloid aggregates of the HET-s prion protein are infectious Proc Natl Acad Sci U S A 2002 99 7402 7407 12032295 10.1073/pnas.072199199 Kicka S Silar P PaASK1, a MAPKKK that controls cell degeneration and cell differentiation in Podospora anserina Genetics 2004 166 1241 1252 15082544 10.1534/genetics.166.3.1241 Laville EY Etude des mécanismes de deux variations contagieuses chez le Nectria haematococca Berck et Br PhD Thesis Orsay: Paris Sud 1971 Daboussi-Bareyre MJ Parisot D Nelson PE, Toussoun TA, Cook RJ Nucleocytoplasmic interactions implicated in differentiation in Nectria haematococca The Fusarium 1981 Philadelphia: Pennsylvania State University Press 306 317 Daboussi-Bareyre MJ Laillier-Rousseau D Parisot D Contrôle génétique de deux états différenciés de Nectria haematococca Can J Bot 1979 57 1161 1171 Daboussi-Bareyre MJ Heterokaryosis in Nectria haematococca: complementation between mutants affecting the expression of two differentiated states J Gen Microbiol 1980 116 425 433 Akins RA Lambowitz AM General method for cloning Neurospora crassa nuclear genes by complementation of mutants Mol Cell Biol 1985 5 2272 2278 2942762 Daboussi MJ Loss of a cytoplasmic factor through ethidium bromide and elevated temperature treatments in Nectria haematococca Can J Microbiol 1985 31 104 108 Saupe S Turcq B Begueret J A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain Gene 1995 162 135 139 7557402 10.1016/0378-1119(95)00272-8 Daboussi-Bareyre MJ Contrôle génétique de deux états différenciés chez l'ascomycète Nectria haematococca 1979 PhD Thesis. Orsay: Université Paris Sud – Centre d'Orsay Brown JS Holden DW Insertional mutagenesis of pathogenic fungi Curr Opin Microbiol 1998 1 390 394 10066509 10.1016/S1369-5274(98)80054-4 Scazzocchio C Sdrin N Ong G Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans: I. Characterization of alleles, dominance and complementation studies, and a fine structure map of the uaY–oxpA cluster Genetics 1982 100 185 208 7049832 Arst HN JrScazzocchio C Bennett J, Lasure L Formal genetics and molecular biology of the control of gene expression in Aspergillus nidulans Gene Manipulations in Fungi 1985 Orlando: Academic Press 309 343 Maeda I Kohara Y Yamamoto M Sugimoto A Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi Curr Biol 2001 11 171 176 11231151 10.1016/S0960-9822(01)00052-5 Wickner RB Edskes HK Roberts BT Baxa U Pierce MM Ross ED Brachmann A Prions: proteins as genes and infectious entities Genes Dev 2004 18 470 485 15037545 10.1101/gad.1177104 Lockshon D A heritable structural alteration of the yeast mitochondrion Genetics 2002 161 1425 1435 12196390 Lalucque H Silar P Incomplete penetrance and variable expressivity of a growth defect as a consequence of knocking out two K+ transporters in the euascomycete fungus Podospora anserina Genetics 2004 166 125 133 15020412 10.1534/genetics.166.1.125 Babai-Ahary A Daboussi-Bareyre MJ Parisot D Isolation and genetic analysis of self-sterility and perithecial pigmentation mutants in a homothallic isolate of Nectria haematococca Can J Botany 1982 60 79 84 Bareyre MJ Laillier-Rousseau D Propriétés de nouveaux mutants affectant les variations morphologiques chez le Nectria haematococca (Berk. et Br.) Wr CR Acad Sc Paris, Ser D 1972 274 3614 3615 Daboussi-Bareyre MJ Synthèse et migration de l'information morphogénétique chez le Nectria haematococca (Berck. et Br.) Wr Physiol Veg 1976 14 517 532 Silar P Two new easy to use vectors for transformation Fungal Genet Newslet 1995 42 73 Crowhurst RN Rees-George J Rikkerink EH Templeton MD High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V) Curr Genet 1992 21 463 469 1617735 Ausubel FM Brent R Kingston RE Moore DD Seidman JG Smith JA Strul K eds Current Protocols in Molecular Biology New York: Wiley Interscience 1987 Malardier L Daboussi MJ Julien J Roussel F Scazzocchio C Brygoo Y Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum Gene 1989 78 147 156 2670677 10.1016/0378-1119(89)90322-3 Orbach MJ A cosmid with a HyR marker for fungal library construction and screening Gene 1994 150 159 162 7959044 10.1016/0378-1119(94)90877-X

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==== Front BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-2-301532095010.1186/1741-7015-2-30Research ArticleLoss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model Wangemann Philine [email protected] Erin M [email protected] Beatrice [email protected] Tao [email protected] Sairam V [email protected] Rajanikanth J [email protected] Lee Jun [email protected] Lorraine A [email protected] Susan M [email protected] Ines E [email protected] Eric D [email protected] Daniel C [email protected] Anatomy & Physiology Department, Kansas State University, Manhattan, Kansas, USA2 Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA3 Department Medicine, Renal Division, Emory University, School of Medicine, Atlanta, Georgia, USA2004 20 8 2004 2 30 30 16 4 2004 20 8 2004 Copyright © 2004 Wangemann et al; licensee BioMed Central Ltd.2004Wangemann et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). Methods Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. Results Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. Conclusions These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome. ==== Body Background Pendred syndrome is a relatively common autosomal-recessive disorder characterized by deafness and goiter [1]. The syndrome is caused by mutations of the PDS gene SLC26A4, which codes for the protein pendrin [2]. Deafness is congenital and generally profound although sometimes late in onset and provoked by light head injury. Vestibular dysfunction is uncommon. Goiter is variable and generally develops around puberty [3]. The cause of goiter appears to be an impairment of iodide fixation in the follicular lumen due to a reduced rate of iodide transport across the apical membrane of thyroid gland epithelial cells [4]. A positive perchlorate discharge test and an enlarged vestibular aqueduct appear to be the most reliable clinical signs of Pendred syndrome [5]. Pendrin is an anion exchanger that can transport Cl-, I-, HCO3 - and formate [6,7]. Expression has been found in the inner ear and thyroid gland consistent with the clinical signs of deafness and goiter [2,3,8]. In addition, pendrin expression has been found in the kidney [9], mammary gland [10], uterus [11], testes [12] and placenta [13]. No expression was found in fetal or adult brain, consistent with a peripheral cause of deafness [2,11]. Expression of pendrin mRNA in the inner ear has been found in several places including the cochlea, the vestibular labyrinth and the endolymphatic sac [8]. The precise location of pendrin protein expression, however, has not yet been determined. The variability of deafness in Pendred syndrome and the observation that deafness is sometimes late in onset suggest that pendrin dysfunction may not be the direct cause of deafness. It is conceivable that pendrin dysfunction favors changes in the expression levels of proteins that are critical for the maintenance of the hearing function. Detailed studies aimed at identifying the direct cause of deafness in Pendred syndrome have recently become possible due to the generation of a pendrin-specific polyclonal antibody [9] and the development of Slc26a4-/- mice, which bear a targeted disruption of the mouse Slc26a4 gene [14]. The aim in the present study was to determine the location of pendrin and the cause of deafness in Slc26a4-/- mice. Methods The endocochlear potential and the endolymphatic and perilymphatic K+ concentrations were measured in young adult mice (1–4 month of age) that either have (Slc26a4+/+) or lack (Slc26a4-/-) a functional gene for pendrin [14]. The mouse strain 129Sv/Ev (Taconic, Germantown, NY) was chosen as the source of Slc26a4+/+ controls, since Slc26a4-/- mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. Slc26a4+/+ and Slc26a4-/- were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched Slc26a4+/+ and Slc26a4-/- that were bred in parallel. Expression of key proteins involved in the generation of the endocochlear potential and the transport of K+ was studied using confocal immunocytochemistry and quantitative RT-PCR. K+ secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University. Confocal immunocytochemistry Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl--free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA [donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave (Calbiochem, La Jolla, CA), and viewed by confocal microscopy (LSM 5 Pascal or LSM 510 Meta, Carl Zeiss, Jena or Göttingen, Germany). Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation. Quantitative RT-PCR Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Kidneys and brain were removed and pulverized in liquid N2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula [14]. Total RNA was isolated and residual DNA contamination was removed by enzymatic digestion (RNeasy micro, Qiagen, Valencia, CA). Quality and quantity of 18S rRNA obtained from kidneys and brain were determined (Nano Assay, 2100 Bioanalyzer, Agilent, Palo Alto, CA). Further, the quality of RNA preparations obtained from stria vascularis and spiral ganglia was assessed (Pico Assay, 2100 Bioanalyzer). Real time RT-PCR was performed on RNA obtained from individual animals (OneStep RT-PCR, Qiagen; Smart Cycler, Cepheid, Sunnyvale, CA) in the presence of 0.2× SYBR green I (Molecular Probes). Transcripts of 18S rRNA and mRNA for the K+ channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers (Table 1). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 50 cycles of 1 min at 60°C, 1 min at 72°C, 7s heating to hot-measurement temperature, 13s hot-measurement at 3–5°C below product melting temperature (Table 1) and 1 min at 94°C. Hot-measurements were performed to eliminate the detection of primer-dimers that had melting temperatures between 72 and 75°C [15]. PCR was followed by a melt (60 to 95°C). The generation of a single product of appropriate size was routinely checked by the presence of a single melt peak and by agarose gel-electrophoresis. Product identity was confirmed by sequencing. Table 1 Primers Template Primers Product Length Melting Temperature Hot Measurement Fidelity n 18S gag gtt cga aga cga tca ga (sense) 316 bp 83.2 ± 02°C 78°C 1.89 ± 0.02 17 tcg ctc cac caa cta aga ac (antisense) KCNJ10 tgg tgt ggt gtg gta tct gg (sense) 411 bp 83.2 ± 02°C 78°C 1.86 ± 0.02 15 tga agc agt ttg cct gtc ac (antisense) KCNQ1 ttt gtt cat ccc cat ctc ag (sense) 239 bp 82.5 ± 02°C 79°C 1.85 ± 0.02 17 gtt gct ggg tag gaa gag (antisense) KCNQ4 ccc gga aac cct tct gtg tc (sense) 245 bp 83.2 ± 02°C 80°C 1.87 ± 0.01 17 aaa gat gag cac cag gaa cc (antisense) Template molecules (T) were quantified according to T = P / (F ^Ct) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold [15]. Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be <0.02% by omission of the RT step. In vitro electrophysiology Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded [16]. A Pt-Ir wire microelectrode with a Pt-black tip was positioned 20–30 μm from the apical surface of the epithelium and vibrated at 200–400 Hz by piezo-electric bimorph elements (Applicable Electronics, Forest Dale, MA; ASET version 1.05, Science Wares, East Falmouth, MA). A Pt-black electrode (26-gauge) served as reference in the bath chamber. The signals from the phase-sensitive detectors were digitized (16 bit) at a rate of 0.5 Hz. The output was expressed as current density at the electrode. In situ electrophysiology Animals were anesthetized with inactin (thiobutabarbital sodium salt, 140 mg/kg ip). The endocochlear potential and the endolymphatic [K+] were measured with double-barreled microelectrodes [17]. Measurements were made in the basal turn by a round-window approach through the basilar membrane and in the apical turn after thinning the bone over the stria vascularis and picking a small hole (~30 μm). K+-selective electrodes were calibrated in solutions of constant cation (K+ and Na+) concentration of 150 mM. The minor selectivity of the K+ electrodes for Na+ produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = Vi + S × log ([K+] + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer (Stat Profile M, Nova Biomedical, Waltham, MA). Data are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p < 0.05. Results and Discussion In situ hybridization in the cochlea has suggested that pendrin mRNA is expressed in cells that reside immediately beneath the spiral prominence on the lateral wall of the external sulcus [8]. To determine the location of pendrin protein expression, we performed confocal immunocytochemistry on cryosections prepared from temporal bones of normal (Slc26a4+/+) mice using an established antibody [3]. Staining was absent when the primary antibody was pre-absorbed with the antigenic peptide (data not shown). Strong expression of pendrin was observed not only in outer sulcus epithelial cells, as predicted from in situ hybridization data, but also in root cells, in apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells that are part of stria vascularis (Fig. 1a,1b,1c). The presence of pendrin in spindle-shaped cells suggests that these cells secrete HCO3 - into endolymph. Pendrin-mediated HCO3 - transport has previously been shown in the kidney [9]. HCO3 - in the cochlea may be generated from CO2 by carbonic anhydrase located in strial intermediate cells [18,19]. CO2 may be supplied by the metabolically highly active stria marginal cells. It is conceivable that pendrin dysfunction interrupts HCO3 - secretion and leads to an accumulation of HCO3 - in stria vascularis. Preliminary data (Wangemann et al., unpublished) support a role for pendrin in HCO3 - secretion into endolymph. Figure 1 Protein localization of pendrin, KCNQ1, ZO-1 and F actin in cochlea and vestibular labyrinth of Slc26a4+/+ and Slc26a4-/- mice. a: Overview of cochlea; bar = 100 μm. b-f: Detail of cochlear lateral wall; bar = 10 μm. g: Detail of utricle; bar = 10 μm. h-i: Detail of ampulla; bar = 10 μm. RM, Reissner's membrane; SC, spindle-shaped cells, SMC, strial marginal cells; SV, stria vascularis; SL, spiral ligament; LIM, spiral limbus. BC, basal cells; SP, spiral prominence epithelial cells; RC, root cells; OS, outer sulcus epithelial cells; VHC, vestibular hair cells; VTC, vestibular transitional cells; VDC, vestibular dark cells; arrows, basal cells at the top and bottom of stria vascularis form tight junctions with surface epithelial cells. The extent of pendrin expression in spiral prominence cells and stria vascularis was determined by labeling KCNQ1, a K+ channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner's membrane (Fig. 1c). In situ hybridization in the vestibular labyrinth suggested that pendrin mRNA is expressed in non-sensory cells [8]. Using confocal immunocytochemistry on cryosections, we observed strong expression of the pendrin protein in the apical membrane of vestibular transitional cells in the utricle and ampullae (Fig. 1g,1h,1i). Dual labeling with KCNQ1 demonstrated that pendrin expression was clearly limited to vestibular transitional cells and did not extend to other non-sensory cells such as vestibular dark cells, which were clearly identified by the expression of KCNQ1 and KCNE1 in their apical membranes. The onset of pendrin expression during development of the mouse inner ear has been determined to be embryonic day (ED) 13 [14]. Morphologically detectable differences in the inner ears of Slc26a4+/+ and Slc26a4-/- mice become evident as early as ED 15, when Slc26a4-/- mice start to develop an enlarged endolymphatic space that persists into adulthood [14]. Interestingly, sensory hair cells in the cochlea appear normal until postnatal day (PD) 7 but show clear evidence of degeneration by PD 15 [14]. These observations suggest that the cochlear environment supports the survival of sensory hair cells in spite of the enlargement of the endolymphatic duct. A normal endolymphatic K+ concentration, which is critical for hair cell survival [20], is established at PD 3 [21] and may persist in Slc26a4-/- at least until PD 7. The time period between PD 7 and 15 is the time when the endocochlear potential develops at the onset of hearing [22]. We hypothesized that a lack of a normal endocochlear potential or an alteration of the endolymphatic K+ concentration could account for deafness in Slc26a4-/- mice. Measurements revealed that the endocochlear potential was absent but that the endolymphatic K+ concentration was normal in adult Slc26a4-/- (Fig. 2a). No significant differences between Slc26a4+/+ and Slc26a4-/- mice were found in perilymphatic (Fig. 2a) or plasma K+ concentrations (Slc26a4+/+, 4.9 ± 0.3 mM, n = 6; Slc26a4-/-, 5.1 ± 0.3 mM, n = 6). These observations suggest that a primary event leading to deafness in Slc26a4-/- mice, and potentially in patients suffering from Pendred syndrome, is the loss of the endocochlear potential. Degeneration of hair cells is probably a response to the loss of the endocochlear potential. Figure 2 Potential, K+ concentrations and pigmentation of stria vascularis in Slc26a4+/+ and Slc26a4-/- mice. a: Endocochlear potential and K+ concentrations in endolymph and perilymph at the apex (A) and base (B) of the cochlea. Numbers adjacent to symbols denote number of measurements. b-d: Pigmentation of stria vascularis in Slc26a4+/+ and Slc26a4-/- mice. b: View of stria vascularis through the bony capsule of the cochlea. OW, oval window, RW, round window; arrows, stria vascularis. c-d: Whole-mounts of stria vascularis isolated from age-matched mice. c: Laser-scanning images, bar = 10 μm, d: Quantification of pigmentation based on optical density. The endocochlear potential is generated by stria vascularis in the lateral wall of the cochlea [17,23]. The potential is generated across the basal cell barrier of stria vascularis by the K+ channel KCNJ10 located in intermediate cells [24], which are connected to basal cells by a high density of gap junctions [25]. Marginal cells of stria vascularis, which form the barrier toward endolymph, transport K+ from the intrastrial space into endolymph and keep the K+ concentration low adjacent to the KCNJ10 K+ channel [26]. To determine the cause of the loss of the endocochlear potential in Slc26a4-/- mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential [27,28]. Intermediate cells of stria vascularis were visualized by their pigmentation. Pigmentation was present in stria vascularis of Slc26a4-/- mice (Fig. 2b), which suggests that intermediate cells are present. Interestingly, pigmentation of stria vascularis was much stronger in Slc26a4-/- than in Slc26a4+/+ mice. To determine in greater detail the cause of the loss of the endocochlear potential in Slc26a4-/- mice, we isolated total RNA from stria vascularis and spiral ganglia of young (1–4 month) Slc26a4+/+ and both young and old (~12 month) Slc26a4-/- mice, assessed amounts of isolated tissues by quantification of 18S rRNA, and quantified the expression of KCNJ10 mRNA. Quantities of stria vascularis isolated from these different mice were similar, since no significant differences were found in the numbers of 18S rRNA molecules (log(rRNA) = 9.46 ± 0.08, n = 17, pooled data). In contrast, quantities of spiral ganglia isolated from young and old Slc26a4-/- mice (log(rRNA) = 9.04 ± 0.18, n = 4 and 9.29 ± 0.15, n = 6) were significantly smaller than in Slc26a4+/+ mice (log(rRNA) = 9.48 ± 0.19, n = 7), consistent with morphometric data (see below). Expression of KCNJ10 mRNA was normal in stria vascularis and spiral ganglia of young Slc26a4-/- mice but significantly reduced in old Slc26a4-/- mice (Fig. 3). Quantifications of KCNQ1 and KCNQ4 mRNA were used to assess possible cross-contamination between the stria vascularis and spiral ganglia preparations based on the assumptions that (1) KCNQ1 is expressed in cells of the stria vascularis but not the spiral ganglia preparation and (2) KCNQ4 is expressed in cells of the spiral ganglia but not the stria vascularis preparation. The number of KCNQ1 mRNA molecules in stria vascularis was 24-fold greater than in spiral ganglia, and the number of KCNQ4 mRNA molecules in spiral ganglia was 5-fold greater than in stria vascularis. These observations validate our measurements of KCNJ10 and KCNQ1 by demonstrating that the microdissected preparations of stria vascularis and spiral ganglia were 98% and 78% pure, respectively. Figure 3 Quantification of KCNJ10 and KCNQ1 mRNA expression in stria vascularis and spiral ganglia of Slc26a4+/+ and Slc26a4-/- mice. a: Electropherogram of total RNA isolated from stria vascularis microdissected from one mouse. The amount of total RNA was obtained from the total integral (shaded) and the amount of 18S rRNA was obtained from the integral of the 18S peak. Sharp peaks representing 18S and 28S rRNA demonstrate the high quality of RNA. Insert: Genotype of Slc26a4-/- mice was verified by the observation of one or few very large rhomboedric otoconia in the utricular macula (arrow). A, crista ampullaris; U, utricular macula. Scale bar: 100 μm. b: Example of real-time RT-PCR data used for quantification of 18S, KCNJ10, KCNQ1 and KCNQ4. Known quantities of 18S rRNA were used to calibrate the threshold. SV, stria vascularis; SG, spiral ganglia. c: Quantification of KCNJ10 and KCNQ1 mRNA in young Slc26a4+/+ and young and old Slc26a4-/- mice. The presence of KCNJ10 mRNA in stria vascularis of Slc26a4-/- mice supports the finding that intermediate cells are present. Expression of the KCNJ10 protein was assessed in young Slc26a4-/- mice by confocal immunocytochemistry. Interestingly, expression of the KCNJ10 protein was absent in stria vascularis but normal in spiral ganglia (Fig. 4). The absence of the KCNJ10 K+ channel in stria vascularis is sufficient to explain the loss of the endocochlear potential [17]. Figure 4 Protein localization of KCNJ10 in the cochlea of Slc26a4+/+ and Slc26a4-/- mice. a: Overview of cochlea; bar = 100 μm. Compare to Fig. 1a to note the enlarged scala media and the distended Reissner's membrane. b-c: Detail of lateral wall and spiral ganglia (insert); main bar: 10 μm, insert: 5 μm. Expression of KCNJ10 in Slc26a4-/- mice was absent in stria vascularis but unchanged in spiral ganglion cells. RM, Reissner's membrane, SV, stria vascularis; SP, spiral prominence; SL, spiral ligament; LIM, spiral limbus; SG, spiral ganglion. Histological evaluation of cryosections revealed an enlargement of scala media with a large bulging of Reissner's membrane and an apparent degeneration of the organ of Corti, as described earlier [14]. Tissue height of stria vascularis was normal (Fig. 5), consistent with the similar numbers of 18S rRNA molecules in isolated preparations (see above). The absence of a change in tissue height is consistent with the presence of intermediate cells [28]. Further, we observed an apparent loss of tissue masses in areas that are normally occupied primarily by type I and II fibrocytes. Spiral prominence in Slc26a4-/- mice was less prominent, spiral ligament thinner and spiral limbus flatter (Fig. 5). The observation that tissue masses were lost in the spiral limbus region is consistent with the finding of a reduced number of 18S rRNA molecules in the spiral ganglia preparation (see above). In addition, we observed an apparent degeneration of stria vascularis, including an increase in pigmentation and an irregular pattern of the tight junctions of marginal cells (Fig. 2c, 6,7). Tight junctions were visualized by F-actin expression. Marginal cells appeared to form a continuous layer. Figure 5 Morphometric analysis of cochlear tissue masses in Slc26a4+/+ and Slc26a4-/- mice. a: locations of measurements. Thickness of stria vascularis (SV) was obtained as average of three distance measurements perpendicular to the surface of marginal cells. Thickness of spiral prominence (SP) was measured perpendicular to a tangential line (dashed) that connects the surface of the outer sulcus (OS) with the basal layer of stria vascularis. Thickness of spiral ligament (SL) was measured perpendicular to the tangential line as distance between the surface of spiral prominence and the interface between spiral ligament and bone (B). Thickness of spiral limbus (LIM) was obtained perpendicular to the surface of the bone as a tangential line that touches the inner sulcus (IS) and reaches from the surface of the spiral limbus to the interface between spiral limbus connective tissue and bone. b: Summary. Data from 7–8 animals contributed to each column. Figure 6 Analysis of marginal and basal cell barriers by in Slc26a4-/- mice. Tight junctions were visualized by F actin. Whole-mounts of stria vascularis were viewed either from the basal cell side (a-f) or from the marginal cell side (g-l). Bright field images verify that the same area was viewed from either side (b and h). Colored bright field images were mixed with images of F actin staining to indicate the position of pigment granules (d, j, f and l). Focus was varied to either visualize the marginal cell barrier (SMC, c-d and i-j) or the basal cell barrier (BC, e-f and k-l). Both the marginal cell (e-f) and the basal cell barrier (i-f) appeared to be intact. It was critical for this finding that pigmentation did not block the path of the laser. Blockage of the laser by pigmentation produces the untrue impression of a discontinuous marginal cell barrier (c-d) or basal cell barrier (k-l). Comparison of images is aided by marking a significant area with a star. Bars = 10 μm. Figure 7 Analysis of marginal and basal cell barriers in Slc26a4+/+ and Slc26a4-/- mice. Tight junctions were visualized by F actin in whole-mounts of stria vascularis. Bright field images were taken to evaluate pigmentation (a, d and g). Note the intact marginal cell (b) and basal cell (c) barriers in Slc26a4+/+ mice. Minimal pigmentation of Slc26a4+/+ mice did not compromise F actin localization. Whole-mounts of stria vascularis from Slc26a4-/- mice were viewed either from the basal cell side (e-f) or from the marginal cell side (h-i). Bright field images verify that the same area was viewed from either side (d and g). Focus was varied to either visualize the marginal cell barrier (SMC, b,e and h) or the basal cell barrier (BC, c,f and i). Both the basal cell (f) and the marginal cell (h) barriers appeared to be intact. Blockage of the laser by pigmentation produces the untrue impression of 'holes' in the marginal cell barrier (e) or basal cell barrier (i). Comparison of images is aided by marking three significant areas with colored stars. Bars = 10 μm. Pendrin-expressing surface epithelial cells in the spiral prominence region are located in an area where basal cells, which are interconnected by tight junctions, form additional tight junctions with surface epithelial cells [29]. A discontinuity of this complex junction in Slc26a4-/- mice would explain the absence of the endocochlear potential. To evaluate the presence of this connection, we determined by confocal immunocytochemistry the expression of ZO-1 and of F-actin, which associate with tight junction complexes. ZO-1 and F-actin expression revealed a continuous layer of basal cells, including a junction of basal cells with surface epithelial cells in Slc26a4-/- mice, as observed in normal mice (Fig. 1c,1d,1e,1f,6,7). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise in the basal cell barrier. The observation that endolymphatic and perilymphatic K+ concentrations were normal in Slc26a4-/- mice suggests that stria vascularis was able to secrete K+ in spite of the apparent signs of degeneration. The rate of K+ secretion necessary to maintain endolymphatic K+ concentration, however, may be less than necessary in Slc26a4+/+ mice given the reduced numbers of sensory hair cells, which provide a major pathway for K+ exit from endolymph [30,31]. In order to substantiate the view that stria vascularis in Slc26a4-/- secretes K+, we measured the magnitude of the bumetanide-sensitive current exiting stria vascularis across the apical membrane of strial marginal cells and determined the expression of the proteins KCNQ1, KCNE1 and SLC12A2, which are essential for K+ secretion [20,32,33], and of GJB2 (Cx26), which is thought to contribute to K+ cycling [23]. K+ secretion is known to be sensitive to 10-5 M bumetanide, a loop-diuretic that inhibits the Na+/2Cl-/K+ cotransporter SLC12A2 [26]. Bumetanide-sensitive currents were found in both Slc26a4+/+ and Slc26a4-/- mice although the magnitude of the current was significantly smaller in Slc26a4-/- mice (22 ± 6 μA/cm2, n = 4 versus 14 ± 2 μA/cm2, n = 4). KCNQ1 and KCNE1, subunits of the secretory K+ channel, were co-localized in the apical membrane of strial marginal cells; the Na+/2Cl-/K+ cotransporter SLC12A2, which is located in the basolateral membrane of strial marginal cells, was found in stria vascularis; and the gap junction protein GJB2 was found in spiral ligament of Slc26a4+/+ and Slc26a4-/- mice (Fig. 8). Co-localization of KCNQ1 and KCNE1 proteins was also observed in vestibular dark cells (Fig. 1i). Expression of KCNQ1 protein in stria vascularis of Slc26a4-/- mice is consistent with the finding of KCNQ1 mRNA expression (Fig. 3). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise of K+ secretion by strial marginal cells or a compromise of gap junction mediated K+ cycling. Figure 8 Protein localization of KCNQ1, KCNE1, SLC12A2 and GJB2 in the cochlear lateral wall of Slc26a4+/+ and Slc26a4-/- mice. a-f: bars: 10 μm. SMC, strial marginal cells; SV, stria vascularis; BC, basal cells; SL, spiral ligament. Conclusions We described locations of pendrin expression in the cochlea and vestibular labyrinth and detected normal endolymphatic K+ concentrations in spite of an enlargement of this fluid compartment. We found that Slc26a4-/- mice lack the endocochlear potential because they do not express KCNJ10 protein. Intermediate cells protect stria vascularis by converting CO2 to HCO3 - and by detoxifying free radicals (Fig. 9). CO2 and free radicals are generated by the large numbers of mitochondria in the metabolically highly active strial marginal cells. Intermediate cells employ carbonic anhydrase to convert CO2 to HCO3 - [18,19] and catalase to detoxify free radicals. To protect themselves from free radical damage, intermediate cells generate glutathione and melanin pigment [34-37]. It is conceivable that loss of pendrin, which may secrete HCO3 - into endolymph, results in an accumulation of HCO3 - and an alkalinization of the intrastrial spaces. This extracellular alkalinization may enhance free radical stress, since it may inhibit the uptake of cysteine and thereby limit production of the protective glutathione [38]. Support for the hypothesis of enhanced free radical stress comes from the observed hyperpigmentation in mice lacking pendrin. Strial hyperpigmentation has also been observed in other conditions that are associated with free radical stress, such as acoustic trauma [37]. Alterations in the cytosolic pH in conjunction with free radical stress may lead to the loss of KCNJ10 protein expression in strial intermediate cells. Function and expression of other K+ channels has been shown to be controlled by the cytosolic pH and free radicals, which encode the metabolic state of the cell [39]. Suppression of the KCNJ10 K+ channel in strial intermediate cells, which is essential for the generation of the endocochlear potential, is probably the direct cause of deafness in Slc26a4-/- mice and patients suffering from Pendred syndrome. Figure 9 Model for the loss of KCNJ10 in the absence of pendrin expression in stria vascularis. Cys, cysteine, Glu, glutamate, Gly, glycine, CA, carbonic anhydrase, GST, glutathione-S-transferase, GSH, glutathione. Competing interests None declared. Authors' contributions PW designed and coordinated the immunocytochemical, morphometrical and molecular biological experiments. EMI and BA carried out confocal immunocytochemistry on cryosections. SJ and SVJ carried out confocal microscopy on whole-mounts of stria vascularis. SVJ and RJM carried out quantitative RT-PCR. DCM designed and coordinated electrophysiological experiments. TW carried out measurements of the endocochlear potential and the endolymphatic K+ concentration. JHL carried out current measurements on strial marginal cells. SMW, LAE, IER and EDG provided the mice and the pendrin-specific antibody. PW and DCM conceived the study. PW wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This work was supported by NIH-R01-DC01098 (PW), NIH-R01-DC00212 (DCM), NIH-R01-DK52935 (SMW) and Core facilities funded by NIH-P20-RR017686 (Confocal Microfluorometry and Microscopy Core, Molecular Biology Core) are gratefully acknowledged. ==== Refs Pendred V Deaf-mutism and goitre Lancet 1896 11 532 10.1016/S0140-6736(01)74403-0 Everett LA Glaser B Beck JC Idol JR Buchs A Heyman M Adawi F Hazani E Nassir E Baxevanis AD Sheffield VC Green ED Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS) Nat Genet 1997 17 411 422 9398842 10.1038/ng1297-411 Royaux IE Suzuki K Mori A Katoh R Everett LA Kohn LD Green ED Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells Endocrinology 2000 141 839 845 10650967 10.1210/en.141.2.839 Bidart JM Mian C Lazar V Russo D Filetti S Caillou B Schlumberger M Expression of pendrin and the Pendred syndrome (PDS) gene in human thyroid tissues J Clin Endocrinol Metab 2000 85 2028 2033 10843192 10.1210/jc.85.5.2028 Reardon W OMahoney CF Trembath R Jan H Phelps PD Enlarged vestibular aqueduct: a radiological marker of Pendred syndrome, and mutation of the PDS gene QJM 2000 93 99 104 10700480 10.1093/qjmed/93.2.99 Scott DA Wang R Kreman TM Sheffield VC Karniski LP The Pendred syndrome gene encodes a chloride-iodide transport protein Nat Genet 1999 21 440 443 10192399 10.1038/7783 Scott DA Karniski LP Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange Am J Physiol Cell Physiol 2000 278 C207 C211 10644529 Everett LA Morsli H Wu DK Green ED Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear Proc Natl Acad Sci U S A 1999 96 9727 9732 10449762 10.1073/pnas.96.17.9727 Royaux IE Wall SM Karniski LP Everett LA Suzuki K Knepper MA Green ED Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion Proc Natl Acad Sci U S A 2001 98 4221 4226 11274445 10.1073/pnas.071516798 Rillema JA Hill MA Prolactin regulation of the pendrin-iodide transporter in the mammary gland Am J Physiol Endocrinol Metab 2003 284 E25 E28 12388138 Suzuki K Royaux IE Everett LA Mori-Aoki A Suzuki S Nakamura K Sakai T Katoh R Toda S Green ED Kohn LD Expression of PDS/Pds, the Pendred syndrome gene, in endometrium J Clin Endocrinol Metab 2002 87 938 941 11836344 10.1210/jc.87.2.938 Lacroix L Mian C Caillou B Talbot M Filetti S Schlumberger M Bidart JM Na+/I- symporter and Pendred syndrome gene and protein expressions in human extra-thyroidal tissues Eur J Endocrinol 2001 144 297 302 11248751 Bidart JM Lacroix L Evain-Brion D Caillou B Lazar V Frydman R Bellet D Filetti S Schlumberger M Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells J Clin Endocrinol Metab 2000 85 4367 4372 11095481 10.1210/jc.85.11.4367 Everett LA Belyantseva IA Noben-Trauth K Cantos R Chen A Thakkar SI Hoogstraten-Miller SL Kachar B Wu DK Green ED Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome Hum Mol Genet 2001 10 153 161 11152663 10.1093/hmg/10.2.153 Pfaffl M Meyer HH Sauerwein H Quantification of insulin-like growth factor-1 (IGF-1) mRNA: development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction Exp Clin Endocrinol Diabetes 1998 106 506 513 10079033 Lee JH Chiba T Marcus DC P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells J Neurosci 2001 21 9168 9174 11717350 Marcus DC Wu T Wangemann P Kofuji P KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential Am J Physiol Cell Physiol 2002 282 C403 C407 11788352 Okamura HO Sugai N Suzuki K Ohtani I Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique Histochem Cell Biol 1996 106 425 430 8911971 10.1007/s004180050060 Lim DJ Karabinas C Trune DR Histochemical localization of carbonic anhydrase in the inner ear Am J Otolaryngol 1983 4 33 42 6424489 Vetter DE Mann JR Wangemann P Liu Z McLaughlin KJ Lesage F Marcus DC Lazdunski M Heinemann SF Barhanin J Inner ear defects induced by null mutation of isk gene Neuron 1996 17 1251 1264 8982171 10.1016/S0896-6273(00)80255-X Anniko M Nordemar H Embryogenesis of the inner ear. IV. Post-natal maturation of the secretory epithelia of the inner ear in correlation with the elemental composition in the endolymphatic space Arch Otorhinolaryngol 1980 229 281 288 6970572 Sadanaga M Morimitsu T Development of endocochlear potential and its negative component in mouse cochlea Hear Res 1995 89 155 161 8600121 10.1016/0378-5955(95)00133-X Wangemann P K+ cycling and the endocochlear potential Hear Res 2002 165 1 9 12031509 10.1016/S0378-5955(02)00279-4 Takeuchi S Ando M Voltage-dependent outward K+ current in intermediate cell of stria vascularis of gerbil cochlea Am J Physiol 1999 277 C91 C99 10409112 Takeuchi S Ando M Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils Cell Tissue Res 1998 293 271 275 9662649 10.1007/s004410051118 Wangemann P Liu J Marcus DC Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro Hear Res 1995 84 19 29 7642451 10.1016/0378-5955(95)00009-S Schrott A Melichar I Popelár J Syka J Deterioration of hearing function in mice with neural crest defect Hear Res 1990 46 1 8 2380119 10.1016/0378-5955(90)90134-B Carlisle L Steel K Forge A Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant Cell Tissue Res 1990 262 329 337 2076537 Luciano L Reiss G Reale E The junctions of the spindle-shaped cells of the stria vascularis: A link that completes the barrier between perilymph and endolymph Hear Res 1995 85 199 209 7559175 10.1016/0378-5955(95)00047-8 Johnstone BM Patuzzi R Syka J Sykova E Stimulus-related potassium changes in the organ of Corti of guinea-pig J Physiol (Lond) 1989 408 77 92 2778743 Zidanic M Brownell WE Fine structure of the intracochlear potential field. I. The silent current Biophys J 1990 57 1253 1268 2393707 Casimiro MC Knollmann BC Ebert SN Vary JC JrGreene AE Franz MR Grinberg A Huang SP Pfeifer K Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange-Nielsen Syndrome Proc Natl Acad Sci U S A 2001 98 2526 2531 11226272 10.1073/pnas.041398998 Dixon MJ Gazzard J Chaudhry SS Sampson N Schulte BA Steel KP Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice Hum Mol Genet 1999 8 1579 1584 10401008 10.1093/hmg/8.8.1579 Spector GJ Carr C The ultrastructural cytochemistry of peroxisomes in the guinea pig cochlea: a metabolic hypothesis for the stria vascularis Laryngoscope 1979 89 1 38 87995 Takumi Y Matsubara A Tsuchida S Ottersen OP Shinkawa H Usami S Various glutathione S-transferase isoforms in the rat cochlea Neuroreport 2001 12 1513 1516 11388439 10.1097/00001756-200105250-00042 Usami S Hjelle OP Ottersen OP Differential cellular distribution of glutathione–an endogenous antioxidant–in the guinea pig inner ear Brain Res 1996 743 337 340 9017265 10.1016/S0006-8993(96)01090-6 Gratton MA Wright CG Hyperpigmentation of chinchilla stria vascularis following acoustic trauma Pigment Cell Res 1992 5 30 37 1631019 Koyama Y Kimura Y Hashimoto H Matsuda T Baba A L-lactate inhibits L-cystine/L-glutamate exchange transport and decreases glutathione content in rat cultured astrocytes J Neurosci Res 2000 59 685 691 10686597 10.1002/(SICI)1097-4547(20000301)59:5<685::AID-JNR12>3.3.CO;2-Q Tang XD Santarelli LC Heinemann SH Hoshi T Metabolic regulation of potassium channels Annu Rev Physiol 2004 66 131 159 14977399 10.1146/annurev.physiol.66.041002.142720

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BMC Med. 2004 Aug 20; 2:30

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BMC Med

10.1186/1741-7015-2-30

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==== Front J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-1-151531223210.1186/1742-2094-1-15ResearchSimilar promotion of Aβ1-42 fibrillogenesis by native apolipoprotein E ε3 and ε4 isoforms Sweeney David [email protected] Ralph [email protected] Harry [email protected] Jonathan D [email protected] Sam [email protected] Cornell University Medical College, 1300 York Ave, Room A569, New York, NY 10021 USA2 Sir James McCusker Alzheimer's Unit, The University of Western Australia, Perth WA, Australia 60093 Sanders-Brown Institute on Aging, University of Kentucky, Lexington, KY 40508 USA4 Cleveland Clinic, Cleveland, OH 44195 USA5 Farber Institute for Neurosciences Thomas Jefferson University 900 Walnut Street, JHN 467 Philadelphia, PA 19107-5587 USA2004 16 8 2004 1 15 15 7 8 2004 16 8 2004 Copyright © 2004 Sweeney et al; licensee BioMed Central Ltd.2004Sweeney et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The apolipoprotein E ε4 allele contributes to the genetic susceptibility underlying a large proportion (~40–60%) of typical, sporadic Alzheimer disease. Apolipoprotein E deficient mice made transgenic for human apolipoprotein E ε4 accumulate excess cerebral amyloid when compared to similarly prepared mice expressing human apolipoprotein E ε3. Therefore, it is important to search for relevant interactions(s) between apolipoprotein E ε4 and Aβ in order to clarify the biological role for apolipoprotein E ε4 in Alzheimer disease. Using a thioflavine T (ThT)-based assay, we have investigated the effects of native human apolipoprotein E isoforms on the kinetics of Aβ fibrillogenesis. No obvious profibrillogenic activity was detected in Aβ1-40-based assays of any native apolipoprotein E isoform. However, when ThT assays were repeated using Aβ1-42, modest, but statistically significant, profibrillogenic activity was detected in both apolipoprotein E ε3- and apolipoprotein E ε4-containing media and was similar in magnitude for the two isoforms. These data demonstrate that native apolipoprotein E possesses "pathological chaperone"-type activity for Aβ: in other words, the data indicate that a chaperone-like misfolding reaction can occur between native apolipoprotein E and Aβ. However, the equipotent activities of the apolipoprotein E ε3 and ε4 isoforms suggests the possibility that either extended co-incubation of apolipoprotein E and Aβ, or, perhaps, the inclusion in the reaction of other fibrillogenesis-modulation co-factors (such as metal ions, or inflammatory mediators such as reactive oxygen species, α2-macroglobulin, apolipoprotein J, etc.) may be required for modeling in vitro the apolipoprotein E-isoform-specific-regulation of extracellular Aβ accumulation that occurs in vivo. Alternatively, other events, such as differential apolipoprotein E-isoform-mediated clearance of Aβ or of apolipoprotein E/Aβ complexes may underlie apolipoprotein E-isoform-dependent Aβ accumulation. ==== Body Background Genetic-neuropathological correlation indicates that the apolipoprotein E type ε4 isoform specifies increased cerebral [1,2] and cerebrovascular [3] accumulation of amyloid β-protein (Aβ). In addition, the apolipoprotein E ε2 isoform can apparently prevent the expression of clinical Alzheimer-type dementia which is otherwise typically associated with amyloidogenic mutations in the amyloid-β protein precursor [4]. Since the apolipoprotein E ε4 allele contributes to the genetic susceptibility underlying a large proportion (~40–60%) of typical, sporadic Alzheimer disease, it is important to search for relevant interactions(s) between apolipoprotein E ε4 and Aβ in order to clarify the biological role for apolipoprotein E ε4 in Alzheimer disease. Currently proposed mechanisms include differential activities of apolipoprotein E isoforms in modulating Aβ fibrillogenesis [5-7] and/or Aβ clearance [8,9]. Many studies of apolipoprotein E modulation of Aβ fibrillogenesis have utilized denatured apolipoprotein E, purified from the serum of human apolipoprotein E homozgotes following extraction in organic solvents [10]. While providing a convenient source of pure apolipoprotein E protein, this preparation does not represent native apolipoprotein E as it exists in vivo. Using a thioflavine T (ThT)-based assay [11] we have investigated the effects of native human apolipoprotein E isoforms on the kinetics of Aβ fibrillogenesis. Methods Synthetic Aβ1-40 or Aβ1-42 (Keck Foundation Protein Facility, Yale University, New Haven CT) was freshly prepared for each assay at a concentration of 16 mg/ml in distilled, deionized water and diluted with phosphate-buffered saline (PBS) to generate a 5 mg/ml working solution. The "aggregation step" consisted of a reaction mixture containing 8 μl Aβ peptide (1 mg/ml final conc) and 32 μl of either apolipoprotein E ε3- or ε4-containing conditioned medium or control conditioned medium from SV40 empty vector-transfected cells. For the investigation of native apolipoprotein E preparations, apolipoprotein E isoforms were generated in the conditioned medium of stably-transfected SV40-apolipoprotein E ε3-, or SV40-apolipoprotein E ε4-, expressing Chinese hamster ovary (CHO) cells (CHO cells lack detectable endogenous apolipoprotein E; data not shown). All conditioned media were prepared using Dulbecco's minimal essential medium supplemented with 0.2% (wt/vol) bovine serum albumin only (no fetal bovine serum). Apolipoprotein E isoform levels were determined by quantitative immunoblotting of conditioned medium and apolipoprotein E-containing serum standards, the latter having been kindly provided by Dr. Petar Alaupovic of the Oklahoma Medical Research Foundation (Oklahoma City OK). Conditioned medium apolipoprotein E concentrations were then standardized using control medium conditioned by SV40 empty vector-transfected cells as diluent, yielding a final concentration of apolipoprotein E of 14 μg/ml, within the range of that reported in human cerebrospinal fluid. Since the final concentration of Aβ peptide was 1 mg/ml, the Aβ/apolipoprotein E stoichiometry (molar ratio) was ~500:1, suggesting models for the Aβ/apolipoprotein E interaction based either on a "catalytic" "pathological chaperoning" model of apolipoprotein E action on Aβ, or with a "seeding" model of Aβ folding. Detailed biochemical characterization of this native apolipoprotein E preparation has been reported [9]. The "aggregation step" fibrillogenesis reaction [11] was incubated at 37°C until the time of the ThT fluorescence measurement, which was performed from 1 to 7 days later. For the "measurement step" [11], 960 μ1 of 10 μM ThT (Nakarai Chemicals, Kyoto, Japan) in 50 mM phosphate buffer (pH 6.0) was added to the "aggregation step" reaction mixture. Within 30 minutes after addition of ThT, fluorescence was measured with a Millipore Cytofluor (Bedford MA) in each of five successive 200 μl aliquots of the reaction mixture, using an excitation filter of 450 nm and an emission filter of 482 nm, and a temperature of 25°C. In order to standardize the ThT assay in our Laboratory, we performed studies of Aβ fibrillogenesis following 1–7 day incubations of Aβ1-40, either in physiological phosphate buffer alone or in the presence of metal ions (Zn2+, Fe2+, or A13+; 1 mM final conc [12]. ThT fluorescence and ultrastructural features were measured daily (not shown). Profibrillogenic activities of the metal ions tested were in agreement with a published report [12] (e.g., Al3+stimulated ThT fluorescence of Aβ1-40 by 3.6 ± 1.1- to 5.7 ± 1.4-fold; p < 0.01), indicating that our Aβ preparations were capable of metal ion-induced fibrillogenesis. Metals were not present during assessment of profibrillogenic effects of apolipoprotein E isoforms. Results and discussion No obvious profibrillogenic activity was detected in Aβ1-40-based assays of any native apolipoprotein E isoform (Table 1). However, when ThT assays were repeated using Aβ1-42, modest, but statistically significant, profibrillogenic activity was detected in both apolipoprotein E ε3- and apolipoprotein E ε4-containing media and was similar in magnitude for the two isoforms (Table 1). The observation of a profibrillogenic effect of apolipoprotein E specifically for Aβ1-42 has been noted [5] and is of particular interest in light of biophysical and molecular neuropathological evidence suggesting that "long" Aβ peptides ending at positions N-42 or N-43 are apparently crucial for the initiation ("seeding") of Aβ deposition [13]. Table 1 Effects of native apolipoprotein E isoforms on fibrillogenesis of Aβ1-40 and Aβ1-42. Fold-effects represent means ± SEM of the quotients of ThT fluorescence values obtained for each Aβ peptide in the presence of apolipoprotein E-isoform-containing conditioned medium divided by ThT values obtained in the presence of conditioned medium lacking apolipoprotein E, derived from empty vector-transfected cells (n = 5–6). Aβ1-40 1 day co-incubation CHO apolipoprotein E ε3 1.0 ± 0.l-fold N.S. CHO apolipoprotein E ε4 1.0 ± 0.l-fold N.S. 7 day co-incubation CHO apolipoprotein E ε3 1.0 ± 0.l-fold N.S. CHO apolipoprotein E ε4 1.2 ± 0.l-fold N.S. Aβ1-42 4 day co-incubation CHO apolipoprotein E ε3 1.7 ± 0.27-fold p < 0.01 CHO apolipoprotein E ε4 1.6 ± 0.18-fold p < 0.005 7 day co-incubation CHO apolipoprotein E ε3 1.7 ± 0.22-fold p < 0.005 CHO apolipoprotein E ε4 1.8 ± 0.19-fold p < 0.0005 These data demonstrate that native apolipoprotein E possesses "pathological chaperone"-type activity for Aβ: in other words, the data indicate that a chaperone-like misfolding reaction can occur between native apolipoprotein E and Aβ, at least at the concentrations and proportions evaluated herein. However, the equipotent activities of the apolipoprotein E ε3 and ε4 isoforms suggests the possibility that either extended co-incubation of apolipoprotein E and Aβ, or, perhaps, the inclusion in the reaction of other fibrillogenesis-modulation co-factors (such as metal ions, or inflammatory mediators such as reactive oxygen species, α1-antichymotrypsin, heparin sulfate-protcoglycan, non-Aβ component, apolipoprotein J, complement, etc.) may be required for modeling in vitro the apolipoprotein E-isoform-specific-regulation of extracellular Aβ accumulation that occurs in vivo. Alternatively, other events, such as differential apolipoprotein E-isoform-mediated clearance of Aβ or of apolipoprotein E/Aβ complexes [8,9,14] may contribute to apolipoprotein E-isoform-dependent Aβ accumulation. Differential anti-inflammatory activity might also play a role. Further investigation will be required in order to elucidate the precise mechanism(s) which specify how apolipoprotein E ε4 promotes Aβ accumulation in human brain and cerebral vessels in vivo. List of abbreviations ThT, thioflavine T; Aβ, amyloid-β peptide; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary cells. Competing interests None declared. Authors' contributions DS performed all assays, including the ThT assay, which was originated by HL. HL also oversaw the transfer of the assay from his lab to ours. RM prepared standard conditioned media from transfected cells provided by JDS. SG oversaw the project, supported the project as noted below, and wrote the manuscript. Acknowledgements This research was supported by USPHS PPG grant AG10491 to S.G. We thank Jan Naslund and Christer Nordstedt (Karolinska Institute, Stockholm, Sweden) for critical comments and helpful discussion. ==== Refs Schmechel DE Saunders AM Strittmatter WJ Crain BJ Hulette CM Joo SH Pelicak-Vance MA Goldgaber D Roses AD Increased amyloid β-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease Proc Natl Acad Sci USA 1993 90 9649 9653 8415756 Holtzman D Bales K Tenkova T Fagan A Parsadanian M Sartorius L Mackey B Olney J McKeel D Wozniak D Paul S Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease Proc Natl Acad Sci USA 2000 97 2892 2897 10694577 10.1073/pnas.050004797 Greenberg SM Rebeck GW Vonsattel JPG Gomez-Isla Hyman BT Apolipoprotein E ε4 and cerebral hemorrhage associated with amyloid angiopathy Ann Neurol 1995 38 254 259 7654074 St George-Hyslop P Crapper McLachlan D Tuda T Rogaev E Karlinsky H Lippa CF Pollen D Alzheimer's disease and possible gene interaction Science 1994 263 537 8290965 Ma J Yee A Brewer HB Das S Potter H Amyloid-associated proteins α 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer β-protein into filaments Nature 1994 372 92 94 7969426 10.1038/372092a0 Wisniewski T Castano EM Golabek A Vogel T Frangione B Acceleration of Alzheimer's fibril formation by apolipoprotein E in vitro Am J Pathol 1994 145 1030 1035 7977635 Evans KC Berger EP Cho C-G Weisgraber KH Lansbury PT Apolipoprotein E is a kinetic but not a thermodynamic inhibitor of amyloid formation: Implications for the pathogenesis and treatment of Alzheimer's disease Proc Natl Acad Sci USA 1995 92 763 767 7846048 LaDu M Falduto M Manelli A Reardon C Getz C Frail D Isoform-specific binding of apolipoprotein E to β amyloid J Biol Chem 1994 269 23403 23406 8089103 Zhou Z Smith JD Greengard P Gandy S Alzheimer amyloid-β peptide forms denaturant-resistant complex with type ε3 but not type ε4 isoform of native apolipoprotein E Molecular Medicine 1996 2 175 180 8726460 10.1007/s0089460020175 Rail SC Weisgraber KH Mahley RW Isolation and characterization of apolipoprotein E Meth Enzymol 1986 128 273 287 3724506 LeVine H Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: Detection of amyloid aggregation in solution Protein Science 1993 2 404 410 8453378 Mantyh P Ghilardi J Rogers S DeMaster E Allen C Stimson E Maggio J Aluminum, iron and zinc ions promote aggregation of physiological concentration of β-amyloid peptide J Neurochem 1993 61 1171 1174 8360682 Iwatsubo T Odaka Suzuki N Mizusawa H Nukina N Ihara Y Visualization of Aβ 42(43) and Aβ40 in senile plaques with end-specific Af3 monoclonals: Evidence that an initially deposited species is Aβ42(43) Neuron 1994 13 45 53 8043280 10.1016/0896-6273(94)90458-8 Naslund J Thyberg J Tjemberg L Wemstedt C Karlstrom AR Bogdanovic N Gandy S Lannfelt L Terenius L Nordstedt C Characterization of stable complexes involving apolipoprotein E and the amyloid β-peptide in Alzheimer disease brain Neuron 1995 15 219 228 7619525 10.1016/0896-6273(95)90079-9

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J Neuroinflammation. 2004 Aug 16; 1:15

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==== Front BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-291531038910.1186/1471-2121-5-29Research ArticleSubcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex Kluge Christoph [email protected] Thorsten [email protected] Susanne [email protected] Shanti S [email protected] Miriam [email protected] Beatrice [email protected]ß Joachim [email protected] Markus [email protected] Dortje [email protected] Karl-Josef [email protected] Biochemistry and Physiology of Plants – W5, University of Bielefeld, Bielefeld, 33501, Germany2 CNRS, UPR 2355, Institut des Sciences du Végétale, Avenue de la terrasse, Gif Sur Yvette, 91198, France3 Department of Biosciences, H. P. University, Shimla, 171 005, India4 Applied Laser Physics and Laser Spectroscopy – D3, University of Bielefeld, Bielefeld, 33501, Germany2004 13 8 2004 5 29 29 18 5 2004 13 8 2004 Copyright © 2004 Kluge et al; licensee BioMed Central Ltd.2004Kluge et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. Results To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. Conclusions All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1. ==== Body Background Vacuolar H+-ATPases are large multi-heteromeric protein complexes located at endomembranes of all eukaryotic cells. Plant V-ATPase has been identified at the vacuolar and various other endomembranes, and also at the plasma membrane [1-3]. The total molecular mass of V-ATPase is estimated to surpass 700 kDa. V-ATPase pumps protons into membrane-surrounded intracellular compartments at the expense of hydrolysis energy of ATP [4]. V-ATPases share a common structure composed of a ball-like head, a membrane-intrinsic part and connecting stalks similar to ATP-producing F-ATP synthases. Biochemical examinations of the subunit composition revealed that V-ATPases are built from up to 14 subunits. The protein subunits are denominated VHA-A through H for soluble components protruding into the cytoplasm (V1-part), and VHA-a, c, c', c", d and e for membrane-associated subunits (V0-part) [5]. In plants, the molecular characterisation of VHA-subunits is only beginning to include also the genes that were identified recently [6,7]. Following the cloning of cDNA sequences coding for VHA-A, -B, -c, and -E until 1995, VHA-D, -F, -C and -G were cloned from A. thaliana, oat and barley [5,7]. However, the first complete set of subunit sequences only became available with the sequencing of the genome of A. thaliana [9]. The second set was cloned from the halotolerant, facultative CAM plant Mesembryanthemum crystallinum [10], and a third set is now available from rice. A comparative analysis of the genes/ESTs revealed that Arabidopsis and Mesembryanthemum express a similar set of subunits [7]. A detailed analysis of the sequences from both species showed that all examined subunits share the same properties in binding of ATP and conducting protons through the proteolipid ring [10]. An important question concerns the composition of the plant V0-sector since its subunit composition is not resolved yet. VHA-a is the subunit with the highest molecular mass within the V-ATPase and reveals a bipartite structure. As first shown in yeast, VHA-a consists of a 50 kDa hydrophilic N-terminal domain and a hydrophobic, membrane-spanning C-terminus [11-13]. Interestingly, Li and Sze [14] could not observe the VHA-a subunit in functional V-ATPase of oat. Therefore, they suggested a role of VHA-a in assembly of plant V-ATPase. The conclusion is in contrast to results in yeast where site directed mutagenesis had allowed to identify amino acids involved in the mechanism of proton translocation across the membrane. Mutant yeast cells devoid of VHA-a or complemented with modified VHA-a variants exhibited the conditional phenotype of V-ATPase deficiency [15]. Another novel subunit in plants that was only addressed recently is VHA-H. VMA13p being the yeast orthologue of VHA-H was cloned and represents the only crystallized subunit of V-ATPase at present [16]. VHA-H is considered to activate and regulate V-ATPase by functionally coupling ATP hydrolysis to proton flow through the Vo-domain [17]. The aim of the study was to improve the understanding of V-ATPase distribution in plant cells with emphasis on the localization of VHA-a and VHA-H in the V-ATPase structure. Three specific questions were answered using different methodology: (i) Is VHA-a part of the V-ATPase structure? (ii) Where are different subunits localized within plant cells? (iii) Is FRET a suitable method to investigate subunit interaction within the complex, for example between VHA-a and VHA-c, and VHA-H and VHA-B. Results Primary structure of VHA-a From the database dbEST (NCBI, USA) a cDNA fragment from M. crystallinum was identified (AI822404) with similarity to known genes coding for VHA-a in yeast. Using RACE-PCR a full length cDNA was obtained with a size of 2783 bp. Its open reading frame encoded a hypothetical protein of 93.1 kDa. Database search with the program FASTA revealed the highest similarities to the entries At2g21410 (77%), At4g39080 (76 %) and At2g28520 (60 %) from the A. thaliana genomic database and also significant similarity on the amino acid level to the genes Stv1 (34 %) and Vph1 (38 %) from S. cerevisiae. These two latter genes are coding for isoforms of the yeast 100 kDa subunit whereas all other subunits of the yeast vacuolar ATPase are encoded by one gene each. The sequence alignment between the newly cloned amino acid sequence from M. crystallinum, the three isogenes from A. thaliana and the two isogenes from yeast (Fig. 1) indicates a structure with two distinct segments distinguished by their degree of similarity: the N-terminus with a low degree of sequence conservation (amino acid 1 to 400) and the C-terminus (amino acid 401 to 816) with a higher degree of conservation between the sequences. This corresponds to the above mentioned domain structure of S. cerevisiae VHA-a with a cytoplasmic and a membrane-integrated domain. The heterogeneity in the N-terminus is characterised through a high number of deletions and insertions most remarkably between the amino acid positions Gly84 to Ile85, and Gly141 to Gln142. In contrast to all other VHA-sequences, in yeast-Stv1 this region contains additional sequence insertions of approximately 20 amino acids. Sequence variation between VHA-a from distinct species and paralogues within same species continues until Glu193. Other domains of the N-terminus vary to a lesser extent. The heterogeneity decreases in direction to the membrane-spanning C-terminus. Figure 1 Amino acid sequence alignment of plant and yeast VHA-a. Comparison of amino acid sequences deduced from the three coding regions detected in the A. thaliana genome, the sequence of M. crystallinum and the two yeast gene products Vph1 and Stv1 using ClustalW-algorithm. Identical amino acids are marked in dark grey, those similar with M. crystallinum in light grey. The programs JPRED (EMBL, Hinxton) and THMM (EBS, Denmark) were used to predict transmembrane domains (labelled with +). Amino acids identified as essential for H+-pumping in yeast are marked with *. The N-terminal amino acids of VHA-a that are conserved in all species are characterised by an above average portion of amino acids with an acid or aromatic character. A comparison of the first 400 amino acids of VHA-a from 10 different species showed the presence of 34 amino acids conserved throughout all species, 10 of which have acidic and 8 aromatic residues. In all sequences compared the representation of aromatic amino acids is higher than the average. In yeast it was shown that sequence motifs with aromatic amino acid residues are involved in the targeting of proteins [18]. An assignment of VHA-a sequences from A. thaliana and M. crystallinum to the distinct yeast isoforms Vph1 and Stv1, in order to define orthologous genes, was not possible on basis of the amino acid sequence alignment. The comparison of the C-termini revealed a higher degree of sequence conservation interrupted through various short insertions or deletions. The sequence was analysed for secondary structures (Predict Protein, EMBL, Heidelberg) and membrane topology (THHM, Denmark). The prediction correlated regions of high sequence conservation with the localisation of putative transmembrane domains (Fig. 1, marked with +). Furthermore, these hypothetical transmembrane domains are in accordance with the membrane-topology suggested by Leng et al. [19] for the yeast Vph1 protein. Mutations in single amino acids of Vph1 have previously allowed the identification of 5 charged amino acids in the membrane spanning helices of the C-terminus whose mutation strongly affected (Lys734, His743, Glu789 and Arg799) or fully inhibited (Arg735) transmembrane H+-transport, although they did not affect the assembly of the vacuolar ATPase [19-23]. These functional amino acids are also conserved in Mc-VHA-a. Detection of VHA-a in membrane preparations in vitro For further examination of McVHA-a, antisera were raised against two polypeptide fragments, i.e. a N-terminal 42 kDa polypeptide and a 13 kDa domain located between predicted transmembrane helices 3 and 4. Both were expressed heterologously in E. coli using the primer combination a-nterm-f and a-nterm-r, and a-memb-f and a-memb-r, respectively. The derived PCR-products were cloned into the expression vector pCRT7-NT (Invitrogen, Holland), introduced and expressed in E. coli. The 42 kDa and 13 kDa polypeptides were purified to homogeneity by chromatography on Ni-NTA and by preparative SDS-PAGE. The 42 kDa polypeptide was recognized by a purified antibody against yeast Vph1 [13] (not shown). The antiserum produced in guinea pig reacted with the heterologously expressed protein aN-term (not shown). The presence of VHA-a was investigated in plant endomembranes and soluble fractions rapidly prepared from 5 week old M. crystallinum plants. In the soluble and the membrane-fraction, the serum against McVHA-aN-term identified two polypeptides with apparent molecular masses between 65 and 70 kDa (Fig. 2). After increasing the NaCl concentration to 500 mM, the apparently full size 95 kDa band was detected in the membrane fraction corresponding to the expected size of untruncated McVHA-a (Fig. 2). To test whether the addition of NaCl to the membrane preparation affected the purification efficiency of other V-ATPase subunits from the V1-sector, membranes and soluble fractions purified with buffer containing either 100 or 500 mM NaCl were also reacted with antibodies against VHA-E [24] and anti VHA-Di recognizing VHA-B and Di [25]. The latter authors demonstrated that this antibody marked both VHA-A and -B. The increased NaCl concentration had no effect on the labelling strength of VHA-E and VHA-A/B (not shown). Interestingly, also McVHA-A, -B and -E were detected both in the membrane and soluble fractions. Figure 2 Immunodetection of VHA-a in membrane and soluble fractions of M. crystallinum leaves. Membranes were isolated in the presence of 100 (1, 2), 500 mM NaCl (3, 4), and without (5) or with (6) protease inhibitor complete®, respectively. Membranes and soluble fraction were separated rapidly for (1)–(4) whereas the standard membrane isolation procedure with ultracentrifugation was undertaken for (5) and (6). The 95 kDa band is seen as the dominant protein in the membrane fraction isolated at high salt and to some part in the isolate obtained in the presence of protease inhibitor. To examine the sensitivity of VHA-a against proteases, membranes were isolated in second approach, in the absence and presence of a protease inhibitor cocktail (complete®, Roche, Mannheim, Germany). Without protection from proteolysis, a band at about 50 kDa and a doublet band above 60 kDa were predominant. In the sample with protease inhibitor, the 95 kDa band appeared although as one of five bands of similar intensity if decreasing transfer efficiency of high molecular mass polypeptides from the gel to the membrane is assumed. Apparently, VHA-a is prone to degradation. However, the results also indicate that VHA-a is part of V-ATPase. To further prove that tentative conclusion, immunoprecipitation was performed using anti VHA-aN-termand anti VHA-amemb followed by Western blot analysis with anti VHA-E and anti-VHA-A (Fig. 3). Each control, i.e. immunoprecipitation without serum and with preimmune serum, gave the expected results of no response (Lanes 1 and 2 in Fig. 3A,3B,3C). With serum, the 55 kDa band of VHA-A and the dimer of VHA-E was seen. Apparently, immunoprecipitation with antibody directed against the N-terminus as well as the membrane part of VHA-a pulled down a complex also containing subunits of the V1 section, and thus probably the holocomplex. Figure 3 Co-immunoprecipitation of VHA-E and VHA-A with VHA-a. Tonoplast enriched membranes of M. crystallinum were solubilized in buffer supplemented with 2% (v/v) Triton X-100. Antibody directed against VHA-a, either the N-terminal or membrane part, was added and immunoprecipitation was performed. The pellet samples and part of the supernatant (10–15% of total) were loaded on a SDS-gel, and Western blot was performed with anti VHA-E or A. The band intensities related to loaded sample size indicate that only a fraction of total V-ATPase was immuno-precipitated with the anti-VHA-a antibodies. As controls, immunoprecipitation was performed without added serum and with preimmune serum. With anti-E, monomeric and dimeric band of 30 and 60 kDa was detected by Western blotting in the precipitate obtained with anti VHA-aN-term, with anti VHA-A, a 65 kDa band was labelled in separations of both, the precipitates obtained with anti VHA-aN-term and anti-VHA-aMemb, respectively. Immunochemical analysis of VHA-distribution A more detailed analysis of the distribution of the VHA-subunits in the plant cell was performed by immuno-labelling of maize root tip cells. To highlight the distribution of the different VHA-subunits in the cell, squashed maize root cells were incubated with anti VHA-A, anti VHA-E or anti VHA-aN-term. Fig 4 shows the staining patterns of the antibodies in young cells devoid of large vacuoles (Fig. 4A,4D,4G), cells with beginning vacuolization (Fig. 4B,4E,4H) and older cells with many vacuoles of 2–5 μm diameter (Fig. 4C,4F,4I). In cells devoid of large vacuoles, anti VHA-E as well as anti-VHA-A marked punctuated compartments whereas in cells with developed vacuoles a nearly complete staining of the tonoplast and an unsteady staining of other small cellular compartments was observed. In a converse manner, anti-VHA-aN-term did not stain the tonoplast of cells of various vacuolisation state. The staining with anti-VHA-aN-term revealed a distinct reticulate pattern and a staining of the nuclear membrane reminding of ER-labelling. Figure 4 Localization of VHA-subunits in plant cells, using anti-VHA-A, anti-VHA-E and anti-VHA-aNterm-antibody Confocal images represent single images of isolated maize root cells. Immuno-staining was performed on maize root cells. Secondary fluorescently labelled antibodies used were anti-rabbit-FITC (A-F) or anti-guineapig (Cy5). Images were colour coded with Adobe Photoshop. Scalebars are 10 μm. Labelling of tonoplast membranes with anti VHA-A (A to C) and anti VHA-E (D-F) in root cells of increasing age, i.e. non-vacuolized to vacuolized (left to right). Anti-VHA-aN-term labelling of root cells of increasing age (G-I). Note that the staining pattern of VHA-aN-term is distinct from the tonoplast labelling with VHA-A and VHA-E in all cases. A double immuno-labelling-technique [26] was then employed to identify the antibody-marked intracellular compartments in detail. The selected antibodies were directed against the aquaporin γ-Tip located in the tonoplast of the lytic vacuole (anti γ-Tip; [27]) and against marker components of the endoplasmic reticulum (ER) (anti-calreticulin). The results of this immuno-staining are shown in Fig. 5. In cells with developed vacuoles, anti-γ-Tip-labelling of the tonoplast (Fig. 5A) co-localised completely with anti-VHA-A (Fig. 5B,5C). Anti VHA-E labelled similar structures as anti-VHA-A and co-localised also with anti-γ-Tip on the tonoplast (not shown). A co-staining of root cells with anti-VHA-A and the ER-marker anti-calreticulin was then performed. Anti-calreticulin marks a specific ER-network including the nuclear membrane (Fig. 5D,5G,5J). The double labelling with antibodies against VHA-A (Fig. 5E) or VHA-E (Fig. 5H) showed no significant co-localisation of the typical tonoplast staining with the ER-marker. When performing a co-labelling of maize root cells with anti-calreticulin (Fig. 5J) and anti-VHA-aN-term (Fig. 5K) revealed a complete co-labelling of the two markers (Fig. 5L). Figure 5 Co-localization of VHA-subunits with the tonoplast marker γ-Tip and calreticulin, a marker for the ER. Confocal images represent single images of isolated maize root cells which were immuno-probed with antibodies directed against marker polypeptides of the vacuolar membrane (γ-Tip) and endoplasmatic reticulum (calreticulin) and simultaneously treated with anti-VHA-antibodies. Secondary fluorescently labelled antibodies used were anti-rabbit-FITC (A), anti rabbit-Cy3 (B, E, H), anti-mouse-FITC (D, G, J) or anti-guineapig-Cy5 (K). Images were colour coded with Adobe Photoshop. Scalebars are 10 μm. In each row, the immuno-decoration with the marker, with the VHA-subunit specific antibody and the superposition of both is shown. (A-C) Root cell labelled with γ-Tip (A) and VHA-A (B). Note the complete co-localisation of the both markers on the tonoplast of small vacuoles (C). (D-F) Double-staining with calreticulin (D) and VHA-A. (G-I) Double-labelling of a root cell with calreticulin (G) and VHA-E (H) reveals a similar result as with VHA-A. Tonoplast labelling and ER-staining are distinct. (J-L) Co-labelling with calreticulin (J) and VHA-aN-term reveals a complete co-localisation of the two signals on the ER. For high resolution, immunogold labelling with anti VHA-aN-term and anti VHA-A was performed on ultra-thin cross sections of maize root cells (Fig. 6). With anti VHA-a pronounced label with gold particles was detected in ER membranes (Fig. 6D), and occasionally a weak labelling of the Golgi apparatus (Fig. 6C). A labelling of tonoplast membranes was not found, indicating that VHA-aN-term is predominantly located on the ER. For comparison, immuno-gold analysis of ultrathin cross sections with anti-VHA-A. revealed labelling of the tonoplast (not shown) and a labelling of the Golgi apparatus significantly stronger than with anti-VHA-a (Fig. 6A). Figure 6 Immunogold-based localisation of VHA-a. Ultra-thin cross sections of maize root cells in 1 mm distance to the tip were decorated anti VHA- (A), premmune serum, (B), anti VHA-aN-term-antibody (C,D), respectively. Sections were washed, treated with secondary antibody linked to 15 nm gold particles and visualized in an electron microscope. Fluorescence resonance energy transfer between VHA-subunits in vivo FRET allows to investigate protein-protein interaction in vitro and in vivo. Both partners have to carry fluorescent labels with overlapping emission (donor fluorophore) and excitation spectra (acceptor) and need to be situated in close proximity. Half maximum energy transfer takes place at distance of the Förster radius R0. Cyan and yellow fluorescent proteins constitute such a FRET pair and were fused to the C-termini of various subunits of V-ATPase. Under the assumption of freely rotating fluorophores, R0 is close to 5 nm. The size of the V-ATPase complex is about 15 nm (diameter) × 25 nm (length from lumen side to tip of head). Arabidopsis protoplasts were co-transformed with vector constructs of VHA-a fused to YFP and VHA-c fused to CFP under the control of the 35S promotor. Upon excitation of doubly labelled protoplasts at 458 nm both, CFP and YFP showed strong fluorescence (Fig. 7A). Fluorescence emission spectra were recorded using a double dichroic mirror which exhibits high reflectivity at ~514 nm. Therefore, the two emission maxima were separated by a minimum (Fig. 7C). This fact renders the quantitative analysis of the FRET efficiency due to the decrease in donor and increase in acceptor fluorescence more difficult. Alternatively, the method of acceptor bleaching can be used to verify energy transfer [28] and can be seen in Fig. 7B, the fluorescence intensity of YFP decreased rapidly within 10 scan cycles upon excitation at 514 nm due to photobleaching to a residual intensity attributable to auto-fluorescence. Simultaneously, the donor fluorescence increased substantially, thus providing direct evidence for energy transfer from CFP to YFP. From the increase in CFP fluorescence upon acceptor bleaching an overall FRET efficiency of ~0.45 is estimated. Assuming freely rotating fluorophores (K2 = 2/3) this corresponds to a distance between VHA-a, and VHA-c of ~5.4 to 7.2 nm (mean 6.3 ± 0.8 nm). Here it has to be pointed out that each ring of the rotor V0 contains 5 VHA-c-subunits and one VHA-c"-subunit. Hence, dependent on the position of the VHA-c/CFP-subunits in the ring different distances between CFP-labelled VHA-c subunits and the YFP-labelled VHA-a subunit will result. Figure 7 FRET between VHA-subunits co-expressed in protoplasts of A. thaliana and onion epidermis cells. (A) Mesophyll protoplasts of A. thaliana were simultaneously transformed with p35S::VHA-a/YFP and p35S::VHA-c/CFP using the polyethylene glycol method. After 20 h, fluorescence emission from protoplasts was measured following excitation at 458 nm and 514 nm, and image analysis in the range of 470 – 500 nm for CFP and 560 – 585 nm for YFP fluorescence, respectively. (B) Acceptor bleaching in dependence on scan numbers. For this experiment, protoplasts expressing VHA-a-YFP were excited at 514 nm and emission was recorded between 550 and 600 nm. (C) Emission spectra of VHA-c-CFP and VHA-a-YFP before (solid line) and after (broken line) acceptor bleaching. (D) FRET between VHA-A-YFP and VHA-B-CFP after co-expression in onion epidermis cells. Excitation was achieved at 458 nm, and 2D emission images were recorded in the range of 470 to 510 nm (CFP), and 550 to 600 nm (YFP). Similar experiments were performed with onion epidermis cells (Fig. 7D) co-transformed with VHA-A/YFP and VHA-B/CFP, and VHA-B/CFP and VHA-H/YFP, respectively. This system was employed for two reasons, (i) to confirm and extent the results from protoplasts and (ii) to work in turgescent cells, not previously subjected to a protoplast isolation stress. Emission spectra were recorded before and after photobleaching of acceptor. Decreases in acceptor and increases in donor fluorescence intensities, respectively, were smaller for these pairs of VHA-fusions than for VHA-a-YFP and VHA-c-CFP (shown in Fig. 7C). Nevertheless there was significant FRET in the case of VHA-A/YFP and VHA-B/CFP and some indication of FRET in the case of VHA-B/CFP and VHA-H/YFP, whereas the co-transformed pair of VHA-A/CFP and VHA-H/YFP gave no FRET. It should be noted that in addition to the highly expressed fusion proteins, untagged endogenous subunits still are present in the cell. Under such condition, formation of partial subcomplexes with possibly varied FRET properties may not be ruled out. Discussion The set of subunits that assemble plant V-ATPase has recently been completed by similarity searches in plant genomic and EST sequences using information on yeast VMA and other orthologues [5,6]. The presence of a subunit of about 100 kDa in the functionally active plant vacuolar ATPase has been under discussion for a long time [14]. Here, for the first time, a cDNA coding for a plant VHA-a was cloned and characterised. McVHA-a as well as the homologous Arabidopsis gene products contain all charged amino acids that have been shown to be essential for proton-translocation at conserved positions in the membrane spanning region of the C-terminus (Fig. 8). Additional evidence for an essential function of VHA-a in V-ATPase was first obtained for V-ATPase of Bos bovis. The specific V-ATPase-inhibitor bafilomycin [29] was shown to bind to the 100 kDa VHA-a subunit and not, as previously suggested to the proteolipid VHA-c. The results were confirmed for all tested species [30]. Bafilomycin also is a potent inhibitor of plant V-ATPase and is routinely used to distinguish V-ATPase-dependent ATP hydrolysis from background activity [31]. In 1999, Li and Sze [14] detected two unknown polypeptides with apparent molecular masses of 63 and 54 kDa in purified catalytically active V-ATPase but no polypeptide with a molecular mass of about 100 kDa. The authors hypothesized that plant V-ATPase is active in the absence of the 100 kDa subunit. Another explanation for this observation could be the sensitivity of VHA-a to degradation through proteases [32], and the detected unknown polypeptides could result from limited proteolysis of VHA-a, producing subunit-fragments still capable of transporting protons. The immunoblots with soluble fractions and membrane-preparations of M. crystallinum (Fig. 2) incubated with anti VHA-aN-term support this hypothesis. The 65 kDa fragment in the cytoplasmic fraction is likely to derive from a proteolytic processing, releasing the soluble portion of McVHA-a. Accordingly, high NaCl concentration in the purification medium could inhibit involved proteases. The hypthesis is supported by the experiment with protease inhibitor cocktail, where the 95 kDa subunit was detected as band with intermediate intensity, although only among other bands that were immuno-responsive to anti-VHA-a antibody. Thus even the protease inhibitors could not fully suppress proteolysis. Immunoprecipitation with the antibodies anti VHA-aN-term and anti VHA-amemb further proved that both the N-terminal hyrophilic portion and the membrane sector of VHA-a are associated with a complete V-ATPase complex. Figure 8 Membrane topology of plant VHA-a and location of the C-termini of individual subunits based on the FRET data. Based on amino acid sequence analysis and similarity with Vph1 (Leng et al. 1999), the topology of Mc-VHA-a is depicted in (A). The relative location of amino acid residues essential for proton pumping or structure are indicated with boxes. The numbers indicate the amino acid positions. In (B), the results from the FRET experiments are summarized: Asterisks tentatively mark the positions of the C-termini where the GFP variants have been fused to. In plants, immuno-cytochemical examinations of the subcellular localisation of single subunits or holocomplexes (mostly using antibodies directed against VHA-A) have previously indicated a distribution of V-ATPase among nearly all endomembranes of the secretory pathway including the plasmalemma [1-3], [33-35]. These findings were supported by measurement of bafilomycin-sensitive ATPase activity in selectively purified endomembranes of plants [36-38]. In our examination the antisera against VHA-A and -E showed a staining of the tonoplast and to a lesser extent also of the ER and Golgi-Apparatus (data not shown). VHA-A and E are part of the cytoplasmically exposed V1-structure of V-ATPase. The presence of both subunits on all these membranes shows the presence of fully assembled V-ATPase. The presence of the active V-ATPase in plants is not only necessary at the tonoplast, since Matsuoka et al. [38] could show that the activity of the vacuolar ATPase on the ER and the GA is necessary for correct targeting of soluble storage proteins to the vacuole. The presence of active V-ATPase on the prevacuolar compartment was concluded from the acid pH-optimum of enzymes involved in vacuolar transport, for example the activity of the vacuolar sorting receptor BP-80 whose action is strictly pH dependent [21]. For this reason a similar staining pattern of all used anti-VHA antisera would have been anticipated, but our results showed distinct staining patterns of anti-VHA-A and -E on the one hand and anti-VHA-aN-term on the other hand. Immuno-labelling indicates that VHA-aN-term antiserum exclusively labels the ER with some rare association on Golgi stacks. These results are surprising since the sequence features of McVHA-a suggest an essential involvement of VHA-a in proton transport. A possible explanation might be the presence of three different isoforms in A. thaliana and O. sativa which all share the localisation of the charged amino acids responsible for proton-translocation (Fig. 1). Based on our results, we suggest that the isoform of VHA-a recognised by anti VHA a-Nterm (an antibody which was generated against the isoform-specific N-terminus) is exclusively associated with V-ATPase localised on the ER. The hypothesis of a compartment-specific localisation of VHA-a subunits is supported through several findings in plants and yeast. In yeast all known subunits and chaperones of the V-ATPase are encoded by one gene. Only the 100 kDa VHA-a is encoded by two different isoforms (Vph1 and Stv1) [13]. In general the subunit-isogenes of the V-ATPase have a very high degree of similarity within each species [16,39]. In a converse manner, the sequences of the VHA-a isogenes are very heterogenic in S. cerevisiae and A. thaliana (Fig. 1) especially in the cytoplasmic region of the N-terminus [40]. A differential localisation was shown for the two yeast isoforms (Vph1 and Stv1) [12]. A detailed examination through Kawasaki-Nishi et al. [40] showed a localisation of Vph1 on the tonoplast, while Stv1 was detected on the late Golgi-apparatus and the prevacuolar compartment of yeast. By expressing chimeric proteins composed of the N-terminus of Stv1 and the C-terminus of Vph1, and vice versa, the authors were able to show that the N-terminus defines subcellular sorting. Following selective enrichment of the differentially localised complexes, both types of V-ATPases were shown to differ in their stability of the V0/V1-complex and in their coupling efficiency [41]. The N-terminus was responsible for the differential coupling activity, whereas the C-terminus mediated the differential dissociation. In plants, Matsuoka et al. [38] were able to distinguish between two different V-ATPase activities through their differential response to the V-ATPase inhibitor bafilomycin. These V-ATPase activities were localised in distinct membrane fractions of the secretory pathway including the vacuolar compartments. An antibody directed against the V-ATPase holoenzyme revealed significant differences in immuno-staining of endomembrane and vacuolar fractions. These and our results on sequence properties of the different plant VHA-a isoforms and the intracellular localisation of VHA-a support the hypothesis that at least two different V-ATPase activities exist in plants, differing in intracellular localisation and sensitivity to bafilomycin. The target of bafilomycin is VHA-a [29]. Thus, both types of V-ATPases might be distinguished through the presence of different isoforms of VHA-a. Our results from FRET analysis also allows to make some structural assignments: From crystal structure of F-ATP synthase, the C-termini of subunit β, homologous to VHA-A, and subunit α, homologous to VHA-B, are located in close vicinity and oriented to the membrane [42]. Occurrence of FRET between VHA-A/YFP and VHA-B/CFP supports the same structural arrangement in V-ATPase. Following crystalization of isolated yeast VHA-H [16], the structure was fitted in 3D reconstructions of plant V-ATPase based on electron microscopic analysis [43] and suggests localization of the C-terminus of VHA-H to the head structure in proximity to VHA-B. In vivo-FRET in protoplasts expressing VHA-B/CFP and VHA-H-YFP confirms the orientation of the C-termini of VHA-B and H in close vicinity. It should be noted that co-expression of other pairs of chimeric donors and acceptors such as VHA-E/CFP and VHA-c/YFP did not elicit FRET after excitation of CFP (not shown). The assumed location of the C-terminus of VHA-c in the lumen of the endomembrane compartments and the C-terminus of VHA-E most likely in the vicinity of the head is in agreement with the negative result, i.e. the absence of FRET between VHA-E/CFP and VHA-c/YFP. The studies exemplify the suitability of FRET to analyse structural features of V-ATPase in vivo. The efficient but variable FRET in cells expressing VHA-c/CFP and VHA-a/YFP allows two conclusions: First, the C-termini of VHA-c and VHA-a are likely to be located on the same, i.e. luminal, side of the endomembrane compartments supporting the topological model with nine transmembrane-domains of VHA-a in plants as previously suggested for yeast [22,23] (Fig. 8). Second, a significant portion of total VHA-a is located in the neighbourhood of VHA-c. The calculated distance of 5.4 to 7.2 nm between donor and acceptor fluorophore corresponds to the diameter of the proteolipid-ring of the rotor, consisting of 5 subunits of VHA-c and 1 VHA-c", respectively. The results confirm that VHA-a is part of the functional complex and not only involved in V-ATPase assembly. More than half (cf. Fig. 2), and possibly all V-ATPase complexes contain the holopolypeptide of 95 kDa. Conclusions The analysis of the primary structure of plant VHA-a revealed the presence of amino acid residues that are essential for proton pumping in yeast. Employing immuno-co-precipitation and FRET it could be demonstrated that subunits VHA-a and VHA-H are part of the V-ATPase complex of plants. Furtheron it is shown that one distinct VHA-a subunit isoform is localized on the ER. The study also shows the usefulness of FRET to study multisubunit protein structures in vivo and in vitro. Methods Plant growth Mesembryanthemum crystallinum and Arabidopsis thaliana were grown in hydroponics and soil culture, respectively, as described in [10,44]. Growth conditions were 120 μmol quanta m-2 s-1, 60% relative humidity, 20°C, and a daily photoperiod of 12 h duration. Rosette leaves from 3- to 5-week-old Arabidopsis plants were taken for protoplast transfection. Zea mays and Hordeum vulgare were germinated on filter paper in the dark at 25°C for 48 h. Cells were isolated from the first 2 mm of the growing root tip. Onion epidermis was stripped from onion bulbs obtained from a local market. Membrane isolation Leaves (50 g) of M. crystallinum were homogenized in a buffer containing 250 mM sucrose, 50 mM Tris-Cl, pH 8.0, 4 mM ethylenediamine tetraacetic acid (EDTA), 4 mM dithiothreitol and a few crystals of phenylmethylsulfonylfluoride [45]. As indicated in a set of experiments, either NaCl was added at 100 or 500 mM concentration or complete protease inhibitor® cocktail (Roche, Mannheim, Germany) was added throughout the procedure. Following differential sedimentation and gradient centrifugation, tonoplast enriched membranes were recovered from a 30%/35% sucrose interphase, sedimented, frozen in liquid nitrogen and stored at -80°C. Gel electrophoresis and Western blot detection Membrane proteins were separated on 12.5% sodium dodecylsulfate polyacrylamide gels, transferred to nitrocellulose and probed with anti-VHA-E [46], anti-VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany) raised in rabbit or anti-VHA-a raised in guinea pig. Following incubation with primary and secondary antibody conjugated with peroxidase, detection was achieved with the lumilight® system according to the supplier (Roche, Mannheim, Germany). Immunoprecipitation For immunoprecipitation, membranes were solubilised in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% (v/v) Triton X-100, 5 μl anti VHA-a antiserum was added, and the samples were shaken at room temperature for 45 min. Then 150 μl protein A-sepharose equilibrated in the same buffer was added. After 15 min, the suspension was placed on a cushion of 1 ml of 40% sucrose and spun at 10,000 × g for 1 min. The sediment was washed thrice with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1 % (w/v) SDS, and finally once in 125 mM Tris-Cl, pH 6.8. The sediment was boiled in loading buffer and analysed by Western blot using rabbit antisera raised against VHA-E or A. Anti-VHA- a antibody preparation and other antibodies used in this study Two antibodies against specific domains of VHA-a were raised in rabbits, and denominated anti-VHA-aN-term and anti-VHA-aMemb. For both the corresponding cDNA fragments of Mc-VHA-a were amplified by PCR using primer combinations a-nterm-f (ATG CGA TCG GAG CCG ATG CAA) and a-nterm-r (TTC ACC CAA CTC ATC GGT GG) encoding the 42 N-terminally located fragment, and a-memb-f (CTT CCA AAG CCC TTT ATT ATG) and a-memb-r (TCA CTC ATG TCC ACC ATG TCA ATC) encoding the polypeptide loop of about 13 kDa located between transmembrane domain 3 and 4 according to the topological model of Vph1p of S. cerevisiae [47]. The gene fragments were cloned into the vector pCR-T7-NT-Topo (Invitrogen, The Netherlands) and transformed into E. coli JM109. The 6x-his-tagged proteins were expressed, purified by chromatography on Ni-nitrilotriacetate columns, separated by preparative SDS-PAGE, excised as protein bands, eluted and used for immunization (Pineda, Berlin). In addition, antisera against subunits VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany), VHA-E, calreticulin ([48], kindly provided by Andrew Smith, Oxford, UK), Jim 84 ([49] kindly provided by Chris Hawes, Oxford, UK), γ-TIP ([27]; kindly provided by John C Rogers, Washington State University, USA) were used in the co-localization studies. Construction of fusions between VHA subunits and variants of green fluorescence protein (GFP) Mc-VHA-a and -c were cloned into the vectors pECFP/pEYFP (Clontech, Palo Alto, USA) in a site-directed manner after amplification from cDNA [10] using the primers a-ges-BamHI-f (AAA AGG ATC CAT GCG ATC GGA GCC GAT GCA A) and a-ges-NcoI-r (AAA AAC ATG GCC TCT TCT TCT TCA CCA ATC GT), McVHA-c with c-ges-BamHI-f (AAA AGG ATC CAT GTC AAC CGT CTT CAA TGG) and c-ges-NcoI-r (AAA ACC ATG GCT GCC CTT GAC TGT CCA GCT CG). Mc-VHA-A and Mc-VHA-H were cloned as described in [10]. The constructs were introduced into the vector p35SGFP [50], so that the chimeric genes were placed under control of the 35S promoter and the original GFP gene was lost. The same strategy was used to produce Mc-VHA-A, -B and -H gene fusions with variants of GFP. Protoplast isolation and transformation methods Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCI2, 4 mM morpholinoethane sulfonic acid, KOH , phl 5.7) and checked for sufficient intactness in the microscope. In short, 1 mm leaf slices of 3- to 5-week-old Arabidopsis plants were vacuum-infiltrated and cell walls were digested in media containing 1.5 % (w/v) cellulase R10 and 0.4 % (w/v) macerozyme R10. Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCl2, 4 mM morpholinoethane sulfonic acid, KOH, pH 5.7) and checked for sufficient intactness in the microscope. 110 μl PEG-medium (4 % (w/v) polyethylene glycol 4000, 0.2 M mannitol, 0.1 M CaCl2) and 20 μl plasmid DNA (3 μg/μl) were added to 100 μl protoplast suspension. The samples were incubated at room temperature for 15 min and then consecutively diluted with 0.5, 1, 2 and 4 ml W5-medium with 15 min incubation steps in between (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM morpholinoethane sulfonic acid, KOH, pH 5.7). Following 24 h incubation at 25°C, sedimented protoplasts were used for analysis. Cells of onion epidermis were placed on filter paper soaked with one-strength MS basal medium in petri dishes and were transiently transformed with a biolistic approach. Gold particles (1.6 μm, 60 mg/ml) were suspended in 50 % glycerol. 8.33 μl of the suspension were mixed with 8.33 μl plasmid DNA (1 μg/ μl), 8.33 μl 2.5 M CaCl2, 3.33 μl 0.1 M spermidine. Sedimented gold particles were consecutively washed with 70 % and 100 % ethanol and resuspended in 8 μl 100 % ethanol, loaded on a macro carrier for transformation with the Particle Delivery System using a rupture disc of 1100 psi (PDS-1000/He, Biorad, Hercules, USA). The distance between macrocarrier and tissue was 12 cm. The epidermis tissue was incubated for about 20 h at room temperature in the dark prior to analysis. Immuno-fluorescence labelling and image acquisition by confocal laser scanning microscopy (CLSM) Immuno-labelling was performed according to [26]. In brief, cells were fixed in 3.7 % para-formaldehyde (10 mM MgSO4, 10 mM EGTA, 1 × phosphate buffered saline, pH 6.8), washed, permeabilised in 0.5% Triton X-100 and washed again. Following blocking of non-specific binding sites with 1 % bovine serum albumin, primary antibody was added for over night at 4°C. Washed samples were incubated with secondary antibody labelled with Cy3, Cy5 or FITC for 1 h. Double labelling was performed by combined application of primary antibodies from rabbit and guinea pig. Slides were mounted with Citifluor Mounting Medium. Fluorescence analysis was performed with a confocal laser scanning microscope Leica TCS-SP2 (Leica, Heidelberg, Germany) equipped with three lasers and excitation wavelengths of 458, 476, 488, 514, 568 and 633 nm. The double dichroic mirror DD488/543 was used for fluorescein isothiocyanate (FITC), and for Cy5 the triple dichroic mirror TD488/543/633 was used. Background was controlled and photomultiplier voltage (800 V) selected for maximum sensitivity in the linear range. Immunogold-labelling and electron microscopy Cells were fixed in 2.5% glutaraldehyde in EM buffer (50 mM KH2PO4, 50 mM NaH2PO4, pH 7.0) for 45 min, washed with EM buffer and dehydrated with a series of increasing concentration of acetone. Samples were embedded in epoxyresin (Transmit EM, TAAB laboratories equipment, Berkshire, Great Britain), cut into ultra-thin cross-sections of 60–70 nm and immobilized on 200 mesh gold nets. Immuno-decoration was performed with antibody diluted in Tris-buffered saline (TBS, 10 mM bovine serum albumin and 0.05 % (w/v) NaN3) for an hour. Samples were washed five times and incubated with secondary antibody conjugated to 15 nm gold particles. The samples were stained with 0.1 % (w/v) uranyl acetate for 5 s and afterwards with 2 % lead citrate. The samples were analysed with an electron microscope (H500, Hitachi, Japan) at 75 kV. Confocal microscopy of GFP-fusion proteins and FRET-measurement Transformed protoplasts and onion epidermis cells were examined for the localisation of the CFP/YFP-fused proteins using the same CLSM set-up as mentioned above. Autofluorescence of 10 protoplasts, as well as reference spectra of YFP and CFP-derived fluorescence were recorded in the spectral range of 480 to 700 nm, averaged and used for corrections. Excitation was recorded at 458 nm (CFP and FRET) and 514 nm (YFP), respectively. Scan speed was 800 Hz. Acceptor dye was bleached with 100 % laser intensity. Emission spectra were recorded and averaged from 20 transformed protoplasts. For a first estimate of transfer efficiency, a Foerster radius for green fluorescence protein variants of Ro = 5 nm [52] was used to calculate the donor/acceptor distance via the equations E = (ICFP/bleached - ICFP/unbleached)/ICFP/unbleached and R = ((Ro6/E)-Ro6)1/6, where E is the transfer efficiency, and ICFP the fluorescence emission intensity in the CFP peak. Abbreviations CFP: cyan fluorescence protein; CLSM: confocal laser scanning microscope; FRET: (Förster) fluorescence resonance energy transfer; PAGE: polyacrylamide gel electrophoresis; Stv1: VHA-a subunit isogene in yeast; VHA: vacuolar H+-ATPase; Vph1: VHA-a subunit isogene in yeast; YFP: yellow fluorescence protein Authors' contributions CK: VHA-a sequence analysis, immuno-cytochemistry, transient expression systems, preparation of anti VHA-aN-term; TS: co-transfection of protoplasts and epidermis cells, CLSM analysis; SB: immuno-co-localisation and discussion; SS: co-immuno-precipitation; MH: preparation of anti VHA-aMemb; BS-J: immuno-co-localisation and discussion; JR and MS: FRET analysis; DG: transformation and construct design, KJD: project design and supervision. Acknowledgements We are grateful to Dr. Uwe Kahmann for immuno-gold labelling. Technical assistance by Ulrike Windmeier and Martina Holt in preparing membranes and isolating V-ATPase and by Marie-Thérèse Crosniere (plate-forme deimagerie IFR87 "la plante et son environnement", CNRS, Gif sur Yvette, France) for the help with the immunochemistry is gratefully acknowledged. The whole work was performed within the framework of the SFB 613, TP A5. ==== Refs Depta H Holstein SEH Robinson DG Lutzelschwab M Michalke W Membranes markers in highly purified clathrin-coated vesicles from Cucurbita hypocotyls Planta 1991 183 434 442 10.1007/BF00197743 Robinson DG Haschke HP Hinz G Hoh B Maeshima M Marty F Immunological detection of tonoplast polypeptides in the plasma membrane of pea cotyledons Planta 1996 198 95 103 10.1007/BF00197591 Robinson DG Hoppenrath M Oberbeck K Luykx P Ratajczak R Localization of pyrophosphatase and V-ATPase in Chlamydomonas reinhardtii Bot Act 1996 111 108 122 Inoue T Wilkens S Forgac M Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases J Bioenerg Biomemb 2003 35 291 299 10.1023/A:1025720713747 Sze H Schumacher K Muller ML Padmanaban S Taiz L A simple nomenclature for a complex proton pump: VHA genes encode the vacuolar H(+)-ATPase Trends Plant Sci 2002 7 157 161 11950611 10.1016/S1360-1385(02)02240-9 Dietz KJ Tavakoli N Kluge C Mimura T Sharma SS Harris GC Chardonnens AN Golldack D Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level J Exp Bot 2001 363 1969 1980 11559732 10.1093/jexbot/52.363.1969 Kluge C Lahr J Hanitzsch M Bolte S Golldack D Dietz KJ New Insight into the structure and regulation of the plant vacuolar V-ATPase J Bioenerg Biomemb 2003 35 377 388 10.1023/A:1025737117382 Ratajczak R Structure, function and regulation of the plant vacuolar H(+)-translocating ATPase Biochim Biophys Acta 2000 1465 17 36 10748245 10.1016/S0005-2736(00)00129-2 The Arabidopsis Initiative Analysis of the genome sequence of the flowering plant Arabidopsis thaliana Nature 2000 408 796 815 11130711 10.1038/35048692 Kluge C Lamkemeyer P Tavakoli N Golldack D Kandlbinder A Dietz KJ cDNA cloning of 12 subunits of the V-type ATPase from Mesembryanthemum crystallinum and their expression under stress Molecular Memb Biol 2003 20 171 183 10.1080/0968768031000084154 Perin MS Fried VA Stone DK Xie XS Sudhof TC Structure of the 116 kDa polypeptide of the clathrin-coated vesicle/synaptic vesciel proton pump J Biol Chem 1991 266 3877 3881 1704894 Manolson MF Proteau D Preston RA Stenbit A Roberts BT Hoyt MA Preuss D Mulholland J Botstein D Jones EW The Vph1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase J Biol Chem 1992 267 14294 14303 1385813 Manolson MF Wu B Proteau D Taillon BE Roberts BT Hoyt MA Jones EW Stv1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p J Biol Chem 1994 269 14064 14074 7514599 Li X Sze H A 100 kDa polypeptide associates with the V0 membrane sector but not with the active oat vacuolar H(+)-ATPase, suggesting a role in assembly Plant J 1999 17 19 30 10069064 10.1046/j.1365-313X.1999.00345.x Landolt-Marticorena C Williams KM Correa J Chen W Manolson MF Evidence that the NH2 terminus of Vph1p, an integral subunit of the Vo sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector J Biol Chem 2000 275 15449 15457 10747882 10.1074/jbc.M000207200 Sagermann M Matthews BW Cloning, expression and crystallization of VMA13p, an essential subunit of the vacuolar H+-ATPase of Saccharomyces cerevisiae Acta Crystallographica 2000 56 475 477 10815216 10.1107/S0907444900000950 Zhou Z Peng AB Crider BP Anderson P Xie XS Stone DK Recombinant SFD isoforms activate vacuolar proton pumps J Biol Chem 1999 274 15913 15919 10336497 10.1074/jbc.274.22.15913 Cooper AA Stevens TH Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases J Cell Biol 1996 133 529 541 8636229 10.1083/jcb.133.3.529 Leng XH Manolson MF Liu Q Forgac M Site-directed mutagenesis of the 100-kDa subunit (Vph1p) of the yeast vacuolar (H+)-ATPase J Biol Chem 1996 271 22487 22493 8798414 10.1074/jbc.271.37.22487 Kawasaki-Nishi S Nishi T Forgac M Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation Proc Natl Acad Sci USA 2001 98 12397 12402 11592980 10.1073/pnas.221291798 Kirsch T Paris N Butler JM Beevers L Rogers JC Purification and initial characterization of a potential plant vacuolar targeting receptor Proc Natl Acad Sci USA 1994 91 3403 3407 8159760 Leng XH Manolson MF Forgac M Function of the COOH-terminal domain of Vph1p in activity and assembly of the yeast V-ATPase J Biol Chem 1998 273 6717 6723 9506970 10.1074/jbc.273.12.6717 Leng XH Nishi T Forgac M Transmembrane topology of the 100 kDa a subunit (Vph1p) of the yeast vacuolar proton translocating ATPase J Bio Chem 1999 274 14655 14661 10329659 10.1074/jbc.274.21.14655 Dietz KJ Rudloff S Ageorges A Eckerskorn C Fischer K Arbinger B Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis Plant J 1995 8 521 529 7496398 10.1046/j.1365-313X.1995.8040521.x Zhigang A Low R Rausch T Lüttge U Ratajczak R The 32 kDa tonoplast polypeptide Di associated with the V-type H+-ATPase of Mesembryanthemum crystallinum L. in the CAM state: A proteolytically processed subunit B? FEBS Letters 1996 389 314 318 8766723 10.1016/0014-5793(96)00556-X Couchy I Minic Z Laporte J Brown S Satiat-Jeunemaitre B Immunodetection of Rho-like plant proteins with Rac1 and Cdc42Hs antibodies J Exp Bot 1998 49 1647 1659 10.1093/jexbot/49.327.1647 Jauh GY Phillips TE Rogers JC Tonoplast intrinsic protein isoforms as markers for vacuolar functions Plant Cell 1999 10 1867 1882 10521518 10.1105/tpc.11.10.1867 Karpova TS Baumann CT He L Wu X Grammer A Lipsky P Hager GL McNally JG Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser J Microscopy 2003 209 56 70 (Oxford) 10.1046/j.1365-2818.2003.01100.x Zhang J Feng Y Forgac M Proton conduction and bafilomycin binding by the V0 domain of the coated vesicle V-ATPase J Biol Chem 1994 269 23518 23523 8089118 Dröse S Altendorf K Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases J Exp Biol 1997 200 1 8 9023991 Tavakoli N Kluge C Golldack D Mimura T Dietz KJ Reversible redox control of the plant vacuolar H+-ATPase activity is related to disulfide bridge formation not only in subunit A but also in subunit E Plant J 2001 28 51 60 11696186 10.1046/j.1365-313X.2001.01130.x Adachi I Arai H Pimental R Forgac M Proteolysis and orientation on reconstitution of the coated vesicle proton pump J Biol Chem 1990 265 960 966 1967251 Herman EM Lix Su RT Larsen P Hsu H Sze H Vacuolar-type H+-ATPases that are associated with the ER and provacuoles of root tip cells Plant Physiol 1994 106 1313 1324 12232411 Long AR Williams LE Nelson SJ Hall Jl Localization of membrane pyrophosphatase activity in Ricinus communis seedlings J Plant Phys 1995 146 629 638 Oberbeck K Drucker M Robinson DG V-Type ATPase and pyrophosphatase in endomembranes of maize roots J Exp Bot 1994 45 235 244 Chanson A Taiz L Evidence for an ATP-dependent proton pump on the golgi of corn coleoptiles Plant Physiol 1985 78 232 240 16664222 Churchill KA Holaway B Sze H Separation of 2 types of electrogenic H+-pumping ATPases from oat roots Plant Physiol 1983 73 921 928 16663344 Matsuoka K Higuchi T Maeshima ? A vacuolar-type H+-ATPase in a nonvacuolar organelle is required for the sorting of soluble vacuolar protein precursors in tobacco cells Plant Cell 1997 9 533 546 12237363 10.1105/tpc.9.4.533 Nelson N Harvey WR Vacuolar and plasma membrane protonadenosine-triphosphatases Physiol Rev 1999 79 361 385 10221984 Kawasaki-Nishi S Bowers K Nishi T Forgac M Stevens TH The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis J Biol Chem 2001 276 47411 47420 11592965 10.1074/jbc.M108310200 Kane PM Parra KJ Assembly and regulation of the yeast vacuolar H(+)-ATPase J Exp Biol 2000 203 81 87 10600676 Braig K Menz RI Montgomery MG Leslie AGW Walker JE Structure of bovine mitochondrial F1-ATPase inhibited by MgADP and aluminium fluoride Structure 2000 8 567 573 10873854 10.1016/S0969-2126(00)00145-3 Domgall I Venzke D Lüttge U Ratajczak R Böttcher B Three dimensional map of a plant V-ATPase based on electron microscopy J Biol Chem 2002 277 13115 13121 11815621 10.1074/jbc.M112011200 Horling F Lamkemeyer P König J Finkemeier I Baier M Kandlbinder A Dietz KJ Divergent light-, ascorbate- and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis thaliana Plant Physiol 2003 131 317 325 12529539 10.1104/pp.010017 Dietz KJ Heber U Mimura T Modulation of the vacuolar H+-ATPase by adenylates as basis for the transient CO2-dependent acidification of the leaf vacuole upon illumination Biochim Biophys Acta 1998 1373 87 92 9733929 10.1016/S0005-2736(98)00094-7 Betz M Dietz KJ Immunological characterization of 2 dominant tonoplast polypeptides Plant Physiol 1991 97 1294 1301 16668546 Nishi T Forgac M The vacuolar (H+)-ATPases-nature's most versatile proton pumps Nat Rev Mol Cell Biol 2002 3 94 103 11836511 10.1038/nrm729 Crofts AJ Leborgne-Castel N Hillmer S Robinson DG Phillipson B Carlsson LE Ashford DA Denecke J Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow Plant Cell 1999 11 2233 2248 10559446 10.1105/tpc.11.11.2233 Horsley D Coleman J Evams D Crooks K Peart J Satiat-Jeunemaitre B Haws C A monoclonal-antibody, JIM-84, recognizes the golgi-apparatus and plasmamembrane in plant cells J Exp Bot 1993 44 223 229 Davis SJ Vierstra RD Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants Plant Mol Biol 1998 36 521 528 9484447 10.1023/A:1005991617182 Abel S Theologis A Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression Plant J 1994 5 421 427 8180625 10.1111/j.1365-313X.1994.00421.x Gadella TW Jrvan der Krogt GN Bisseling T GFP-based FRET microscopy in living plant cells Trends Plant Sci 1999 4 287 291 10407445 10.1016/S1360-1385(99)01426-0

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==== Front Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-1-21529197210.1186/1742-4682-1-2ResearchA steady state analysis indicates that negative feedback regulation of PTP1B by Akt elicits bistability in insulin-stimulated GLUT4 translocation Giri Lopamudra [email protected] Vivek K [email protected] KV [email protected] Department of Chemical Engineering and School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India2004 3 8 2004 1 2 2 22 6 2004 3 8 2004 Copyright © 2004 Giri et al; licensee BioMed Central Ltd.2004Giri et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The phenomenon of switch-like response to graded input signal is the theme involved in various signaling pathways in living systems. Positive feedback loops or double negative feedback loops embedded with nonlinearity exhibit these switch-like bistable responses. Such feedback regulations exist in insulin signaling pathway as well. Methods In the current manuscript, a steady state analysis of the metabolic insulin-signaling pathway is presented. The threshold concentration of insulin required for glucose transporter GLUT4 translocation was studied with variation in system parameters and component concentrations. The dose response curves of GLUT4 translocation at various concentration of insulin obtained by steady state analysis were quantified in-terms of half saturation constant. Results We show that, insulin-stimulated GLUT4 translocation can operate as a bistable switch, which ensures that GLUT4 settles between two discrete, but mutually exclusive stable steady states. The threshold concentration of insulin required for GLUT4 translocation changes with variation in system parameters and component concentrations, thus providing insights into possible pathological conditions. Conclusion A steady state analysis indicates that negative feedback regulation of phosphatase PTP1B by Akt elicits bistability in insulin-stimulated GLUT4 translocation. The threshold concentration of insulin required for GLUT4 translocation and the corresponding bistable response at different system parameters and component concentrations was compared with reported experimental observations on specific defects in regulation of the system. Insulin signaling pathwayGLUT4TranslocationEnzyme cascadeFeedback loopsBistable switch ==== Body Background In living systems, extracellular information is processed through signal transduction machinery to appropriately regulate cellular function. This information processing machinery is made up of a complex web of enzyme cascades, allosteric interactions and feedback loops. Depending on their regulatory design these signaling networks elicit diverse responses, but display many common operating principles. A recurring theme in signaling systems is switch-like responses to graded or transient input signal. Various mechanisms are known to generate such all-or-none responses [1]. Bistability is one such system level property, in which, the system switches between two discrete stable steady states without being able to rest in an intermediate state. Bistable systems exhibit hysteresis wherein, the value of input stimulus required for system transition from one state to another is quite different from the value required for reverse transition. Both computational and experimental analyses have shown that bistability plays a significant role in cellular differentiation and cell cycle progressions [2-5], production of biochemical memory [6], microbial metabolic systems [7], lateral signal propagation [8] and protein translocations [9]. Existence of bistability in cellular regulation has been attributed to nonlinearity embedded in positive feedback loop or double negative feedback loop [10]. Here, we present steady state simulation results of metabolic insulin signaling pathway comprising of positive feedback loops and show that this system can convert graded inputs into switch-like bistable output response. Insulin is the most potent anabolic peptide hormone known that elicits myriad biological responses by specifically binding to insulin receptor and simultaneously stimulating multiple signaling pathways to regulate growth, differentiation and metabolism. Insulin maintains glucose homeostasis by stimulating the uptake, utilization and storage of glucose in muscle and adipose tissue, and inhibits hepatic glucose production [11]. Defects in any of the pathway components lead to disturbance in growth, differentiation, and in the homeostasis of glucose and lipid levels. This leads to disease conditions such as type 2 diabetes, hypertension, obesity and a cluster of abnormalities characterized by insulin resistance or deficiency. In such a condition, normal circulating concentration of insulin is insufficient to elicit appropriate response [12,13]. Studies over the last century have identified the major insulin signaling components involved in the regulation of glucose uptake into cells and its various defects in diseased states. A wide family of glucose-transporter proteins localized in the plasma membrane, facilitate uptake of glucose from the blood into tissues. Among different isoforms, only glucose transporter isoform-4 (GLUT4) is specifically expressed to promote glucose uptake in insulin sensitive tissues, viz. muscle and adipose, and in response to insulin, GLUT4 gets translocated to the plasma membrane from intracellular vesicles [14]. The biological action of insulin is initiated by binding to the tyrosine kinase receptor and its subsequent activation. The activated tyrosine kinase receptor undergoes autophosphorylation and catalyzes the phosphorylation of several intracellular substrates including the insulin-receptor substrate (IRS) proteins (Fig. 1). The activated IRS isoform-1 protein further activates downstream components to elicit translocation of GLUT4 [11]. There are several downstream kinases like PI-3 kinase, Akt (or protein kinase B) and protein kinase C-ζ (PKC-ζ) demonstrated to be potentially capable of phosphorylating upstream proteins like IRS-1 and tyrosine phosphatase 1B (PTP1B) thus serving as negative and positive feedback loops respectively [15]. Other than feedback loops, crosstalk between multitudes of signal transduction pathways have also been reported, thus making the insulin-signaling pathway a highly intricate network [11]. Figure 1 Simplified representation of molecular mechanism involved in insulin signaling pathway that regulates glucose transporter (GLUT4) translocation to cell membrane. Some of the details like, other isoforms of insulin receptor substrate and multiphosphorylation of insulin receptor substrate are not shown here. Nomenclature: GLUT4: Glucose-transporter isoform 4; IRS-1: Insulin receptor substrate-1; PI3K: Phosphatidylinositol-3-kinase; PI (3, 4, 5) P3: Phosphatidylinositol (PI)-3, 4, 5-tiphosphate; PDK1: phosphosinsositide-dependent kinase 1; Akt: Protein kinase Akt or protein kinase B (PKB); PKC: Protein kinase C-ς; PTP1B: Protein tyrosine phosphatase 1B; PTEN: 3' lipid phosphatase; SHIP2: 5' lipid phosphatase; Detailed description of signaling events are given in the methods section. Letter 'P' indicates phosphorylated species. Although studies on various cell lines, transgenic and knock-out mice, have helped to uncover and characterize the different components involved in insulin signaling pathway, there are many voids in our understanding of the precise molecular mechanisms of signal transduction and cellular effects of insulin [16,17]. The major hurdles are complexity of insulin signaling pathway and technical problems like experimental methodology employed for system level quantification. For example, depending upon different techniques employed, quantification of GLUT4 translocation in response to insulin binding yielded different results in the same cell type [18]. Recent technical developments however have helped in studying the localization and translocation of signaling proteins and overall quantification of signaling processes in single cells has been possible [19]. In such a scenario, it is pertinent to ask questions regarding the design principles involved in intracellular regulation. For example, what does a particular regulatory structure accomplish and how does it help in exhibiting different physiological responses. Based on available experimental data, computational and mathematical analysis can answer some of these questions and possibly propose new experiments and hypotheses. Earlier mathematical modeling studies of insulin signaling pathways have focused on subsystems of the pathway, like insulin receptor binding kinetics [20,21], receptor recycling [22] and GLUT4 translocation [23-25]. Recently a comprehensive dynamic model of metabolic insulin signaling pathway was presented, which involved most of the known signaling components [26]. Although the model correlated well with the published experiment data, authors did not discuss the system level regulatory design of insulin signaling system. In the present work, we have developed a steady state model of insulin signaling to generate dose response curves for fractional translocation of GLUT4 to varying input insulin stimuli. One of the main objectives was to investigate the effect of inherent signaling structure made up of phosphorylation cycles, allosteric interactions and feedback loops on the system level response of insulin on GLUT4 translocation. Furthermore, we were interested in examining whether the regulatory design consisting of positive feedback loops in insulin signaling pathway exhibits bistable response. We solved the steady state equations for the entire metabolic insulin pathway including the positive feedback loops numerically, and found that GLUT4 gets translocated to the plasma membrane in an all-or-none manner in response to a varying concentration of input insulin stimuli. We show that GLUT4 translocation switches between the on-state and off-state and exhibits hysteresis in its response to increasing and decreasing input insulin concentration. This input-output relationship was then studied at various concentration of signaling components and system parameters in order to monitor the range over which this response persisted. We discuss these results by comparing with the known specific defects in regulation of the system (insulin dependent diseases) that lead to improper glucose uptake into the cell. Methods Figure 1 shows a simplified representation of molecular mechanisms involved in insulin signaling pathway. The metabolic insulin-signaling pathway used for the steady state simulation in the present work is shown in Fig. 2. This schematic representation is a compilation of various interactions in insulin pathway which have been very well reviewed [11-27]. We have used the framework of Goldbeter and Koshland [28] to model the insulin system at steady state and accordingly an equivalent rate constant and Michaelis-Menten constant nomenclature scheme is applied. The detailed list of the steady state equations for covalent modification cycles, equilibrium relationships for allosteric interactions, mass balance equations for total species and parameters used in the simulations are provided in Appendix. All component enzyme concentrations are represented with respect to whole cell volume. Most of the kinetic/equilibrium constants are taken from the literature. In this analysis, the reactants like ATP and PPi concentrations are assumed to be constant. In the following paragraphs we present the system considered and assumptions made during the analysis. Figure 2 Schematic representation of metabolic Insulin signaling pathway used for the steady state analysis. Nomenclature: I, Insulin; X, unbound surface insulin receptor; XI, unphosphorylated once-bound surface receptor; XIP, phosphorylated once-bound surface receptor; XI2P, phosphorylated twice-bound surface receptor; Xi represents intracellular receptor pool; XIPi and XI2Pi are internalized form of XIP and XI2P; phosphatase PTP catalyzes the dephosphorylation of AP, XIP, XIPi and XI2Pi. A, unphosphorylated IRS-1; AP, phosphorylated IRS-1; B, inactive PI3-kinase; APB, phosphorylated IRS-1 and PI3-kinase complex; CP3, lipid PI[3,4,5]P3; CP2, lipid PI[4,5]P2; CP2', lipid PI[3,4]P2; phosphatase SHIP2 catalyzes dephosphorylation of CP3 to form CP2', phosphatase PTEN catalyzes dephosphorylation of CP3 to form CP2; F, inactive Akt and PKC-ς; FP, phosphorylated Akt and PKC-ς; E8 dephosphorylates FP; E6 phosphorylates CP2' to form CP3; FP activates GLUT4 from intracellular location to plasma membrane. GC and GM represent GLUT4 in cytoplasm and on plasma membrane respectively. Kd1 to Kd3 are dissociation constants; Kd4 and Kd5 are distribution coefficients; Kmr, Km, Km1to Km8 are Michaelis-Menten constants; k, kp, kd, ks, k0, k1 to k13 are reaction rates as shown in the figure. Insulin initiates its biological action by interacting with the insulin receptor, which belongs to a superfamily of tyrosine kinase receptors. On binding to the first insulin molecule, the receptor gets auto-phosphorylated and is dephosphorylated by phosphatase PTP1B [12]. The phosphorylated insulin receptor can either bind with another insulin molecule or undergoes dissociation. Binding of the second insulin molecule does not affect the phosphorylation state of the receptor. Here we have assumed that the concentration of unbound phosphorylated receptor is negligible. Thus, phosphorylated receptors can exist as species bound to either singly or doubly bound molecules of insulin. Insulin bound phosphorylated receptor rapidly gets internalized into the endosomal apparatus of the cell before it gets dephosphorylated by PTP1B and incorporated into intracellular receptor pool [29]. However recent studies indicate that, PTP1B might interact with insulin receptor directly and deactivate it without internalization [30]. We have assumed that, the membrane bound phosphorylated insulin-receptor and its internalized form, both get dephosphorylated by PTP1B. The rate equation for intracellular receptor at steady state is represented as where kp is rate constant and Kmr is Michaelis-Menten constant for dephosphorylation of internalized insulin receptors XIPi and XI2Pi. The term kd is first order degradation rate constant and ks is zero order synthesis rate constant of intracellular receptor Xi. The receptor exocytosis and endocytosis are assumed to be at quasi-equilibrium because of their faster time scales than the synthesis and degradation of receptors [26]. The phosphorylated active receptors further catalyze phosphorylation of several intracellular substrates including the IRS proteins, GAB-1, Shc and c-Cab1 [16]. Among these, IRS-1 protein is known to participate in the regulation of GLUT4 translocation. In the present study we have assumed that, at steady state the twice-bound phosphorylated receptor catalyses the phosphorylation of IRS-1 protein while neglecting the activation of GAB-1, Shc, c-Cab1. The phosphorylated active IRS-1 further binds and activates PI3 kinase and this association is assumed to occur with a stoichiometry of 1:1. Activated PI3 kinase further phosphorylates phosphatidylinositol-(4,5)-bisphosphate (PI-4,5-P2) to form phosphatidylinositol -3,4,5-triphosphate, (PIP3). The dephosphorylation of PIP3 to form PI-4,5-P2 is catalyzed by phosphatase PTEN, whereas, PIP3 is dephosphorylated to form PI-3,4-P2 by phosphatase SHIP2. Active PIP3 then is known to interact allosterically with phosphosinsositide-dependent kinase 1 (PDK1) and which in turn appears to phosphorylate kinase Akt (or protein kinase B) and protein kinase C-ζ (PKC-ζ) [11]. However, as the interaction due to PDK1 is unclear, active PIP3 is assumed to play a role in phosphorylation of Akt and PKC-ζ. Since the parameters affecting the modification-demodification of Akt and PKC-ζ are considered to be similar, their modification is represented as a single enzyme cascade (Fig. 2). The downstream elements of Akt and PKC-ζ, which effect GLUT4 translocation, are also unknown [11-13]. Therefore, we have assumed that phosphorylated Akt and PKC-ζ directly activate the GLUT4 translocation to the plasma membrane. In the basal state, GLUT4 slowly recycles between the plasma membrane and intracellular vesicular compartment. The phosphorylated Akt and PKC-ζ favor GLUT4 translocation (exocytosis) to the plasma membrane and thus increase glucose uptake as a response to insulin binding to the receptor [14]. Here, total GLUT4 (Gt) is assumed to be sum of GLUT4 concentration in the cytosol (GC) and on the membrane (GM). The rate equation for GLUT4 species in cytoplasm at steady state is represented by, where, k9 is the basal zero order synthesis rate of GLUT4, k10 is basal first order degradation rate, k11 is the insulin-activated GLUT4 exocytosis, k12 and k13 are basal first order rate of exocytosis and endocytosis, respectively. As assumed by Sedaghat, et al. [26], the basal equilibrium distribution of cell surface GLUT4 and GLUT4 in the intracellular pool are taken as 4% and 96%. The insulin signaling pathway has been shown to consist of multiple feedback loops [15]. Active Akt is known to phosphorylate and thereby negatively regulate the upstream phosphatase PTP1B. This phosphorylation impairs the ability of PTP1B to dephosphorylate insulin receptor and IRS-1 by 25% [31]. This represents overall positive feedback loop as Akt inhibits signal attenuation enzyme PTP1B. The resulting circuit also represents a double negative feedback loop, in which phosphorylated protein negatively regulate the phosphatase that dephosphorylates it. To incorporate these feedback loops we assumed that active Akt affects the total active PTP1B enzyme and thus inhibits the dephosphorylation of the receptor and IRS-1. The feedback effect of Akt on PTP1B was incorporated by following relationship where, [PTP]max is maximum PTP1B concentration, PTPt is the total active PTP1B concentration after incorporating the effects of feedback, AktP represents the phosphorylated Akt concentration influencing the PTPase activity, and kf represents the half saturation constant quantifying feedback. The value of kf was estimated based on the assumption that 25% of PTP1B is inactivated by total AktP [31]. Thus, kf is appropriately calculated so that the first term [kf /[kf + AktP]] is equal to 0.75. In absence of feedback effects, PTPt equals PTPmax. The set of equations given in 'appendix' and in 'methods' section were solved numerically using fsolve program of Matlab (The MathWorks Inc. USA). The accuracy of the simulation was verified by numerically checking the mass balance of all species. The steady state modeling of entire insulin signaling was evaluated including the feedback loops and estimating the fractions of GLUT4 translocated to the plasma membrane for a particular concentration of insulin. Thus, the overall action of insulin on GLUT4 translocation is quantified as, where, f is fractional GLUT4 on plasma membrane, GM is GLUT4 concentration on plasma membrane and Gt is total GLUT4 concentration with respect to whole cell volume. Results Bistability in GLUT4 translocation to plasma membrane Fig. 3A shows the predicted dose response curve of steady state fraction of GLUT4 bound to the plasma membrane at different concentrations of insulin. The predicted dose response curve indicates that, there are three steady states exist between 0.01 nM and 0.05 nM of insulin for GLUT4 translocation (curve b, Fig 3A). Out of these three steady states, GLUT4 gets distributed between two discrete stable steady states, either at plasma membrane or in the cytosol without settling in an intermediate unstable state, thus showing a typical hysteresis response. Due to hysteresis, the dose response curve appears to split and we obtain two distinct half-maximal concentrations (K0.5, insulin concentration required for 50% of GLUT4 to reside on the plasma membrane). This represents two threshold concentrations of insulin required for GLUT4 translocation switching on (GLUT4 translocation to plasma membrane at 0.05 nM) and switching off (GLUT4 translocation from to plasma membrane at 0.01 nM). Figure 3 Hysteresis and bistability in insulin-stimulated GLUT4 translocation. A. Dose response curve of insulin stimulated fractional GLUT4 on plasma membrane. Curve 'a' is sigmoidal dose response curve [~Hill coefficient of 3.1] obtained in absence of feedback loop. Curve 'b' represents hysteresis in insulin-stimulated fractional GLUT4 on plasma membrane in presence of feedback loop which impairs the ability of PTPase by 25%. Arrows indicate the switching-on [up arrow] and switching-off [down arrow] GLUT4 translocation. B. A simulated type 2 diabetic condition represented by dose response curve of insulin-stimulated fractional GLUT4 on plasma membrane at higher phosphatase PTP1B concentration. Curve 'a' is typical bistable response obtained in presence of positive feedback loops [PTP1B conc. 0.039 nM]. Curve 'b' represents dose response curve when PTPase concentration was increased by 3 fold [PTP1B conc. 0.098 nM]. A 3-fold increase in the PTPase concentration increased the half-maximal concentration by 100 fold and the response looses bistability. The observed hysteresis is characteristic of a bistable response obtained due to negative feedback regulation of upstream signal attenuation enzyme PTP1B by downstream kinase Akt. Experimental data available in the literature indicates a subsensitive response of insulin, requiring ~130 fold change in insulin concentration for the maximal GLUT4 translocation to plasma membrane [32]. Our results show an ultrasensitive response in insulin-stimulated GLUT4 translocation due to bistability (~4-fold change in insulin concentration); however, the half saturation values match with that of experimental data. The response was ultrasensitive (Hill coefficient ~3.1) and not bistable in absence of feedback loops (curve a, Fig 3A). Effect of system component concentration on GLUT4 translocation To examine the influence of pathological conditions arising due to variations in protein expression levels on final output response of insulin, we varied the concentration of individual signaling components IRS-1, PI3K, lipids, PKC-ζ, Akt and phosphatases, PTP1B, PTEN and SHIP2 over a wide range. For each case, the dose response curve of fractional GLUT4 on the plasma membrane at various insulin concentrations was obtained and the response was quantified in-terms of half saturation constant. To illustrate this, we consider a case of increase in PTP1B concentration. Fig. 3B shows the dose response curves for insulin stimulated GLUT4 translocation at plasma membrane at two different concentrations of PTP1B. At high PTP1B concentration, the bistable dose response curve becomes monostable (but, still ultrasensitive) and shifts to the right. This indicates a nullifying effect of negative feedback regulation on PTP1B by Akt and higher requirement of insulin for maximal translocation of GLUT4. Thus, in Fig 3B curve 'a' and curve 'b' can be characterized by two and one half saturation values respectively. Fig. 4A and 4B show the distinct half saturation constant values obtained for switching-on and switching-off of GLUT4 translocation at various concentrations of IRS-1 and Akt respectively. Such an increase or decrease in the half-maximal concentration of insulin characterizes the decrease and increase in insulin sensitivity found in diseased conditions. The threshold concentration of insulin required for switching-on GLUT4 translocation decreases with increase in IRS-1 concentration. This implies that, increase in IRS-1 concentration amplifies the input signal and beyond a certain concentration of IRS-1 [~3 nM], the system looses bistability. Similar results were obtained for variations in lipid, PI3K and insulin receptor concentration (results not shown). GLUT4 translocation at various concentrations of Akt shows that the system becomes monostable when Akt concentration is decreased. However, the degree of bistability (i.e., difference between half maximal concentrations for switch-on and off) increases with increase in Akt concentration and furthermore, the threshold value to activate GLUT4 translocation decreases. Figure 4 Half-maximal concentration of insulin required for 50% GLUT4 translocation at elevated levels of signaling components. Curve 'a' shows half maximal concentration of insulin required to switch-on GLUT4 translocation. Curve 'b' shows half maximal concentration of insulin required to switch-off GLUT4 translocation. Arrow indicates physiological concentration of particular signaling components. A. Half saturation constant at various concentration of IRS-1. Simulated results indicate increased insulin sensitivity when IRS-1 overexpressed. B. Half saturation constant at various concentration of Akt. Simulated results indicate increased insulin sensitivity when Akt overexpressed and loss of bistability when Akt concentration decreased below 0.01 nM. C. Half saturation constant at various concentration of PTP1B. Simulated results indicate decreased insulin sensitivity when PTP1B overexpressed. D. Half saturation constant at various concentration of PTEN. Simulated results indicate decreased insulin sensitivity when PTEN overexpressed. To study the effect of signal attenuation enzymes such as phosphatases on the output response, the concentrations of PTP1B, PTEN and SHIP2 were altered over a wide range, keeping other parameters constant. Fig. 4C and 4D show the influence of variation in concentrations of PTP1B and PTEN on half saturation constant of insulin. Increase in PTP1B and PTEN concentration results in a drastic increase in the threshold concentration of insulin required to switch-on or switch-off GLUT4 translocation. This illustrates that more insulin than physiological concentration is required at higher phosphatase (PTP1B or PTEN) concentrations to translocate GLUT4 from cytoplasm to plasma membrane. For example, around 16-fold change in the insulin concentration is observed for a 1.5-fold increase in PTP1B concentration from 0.039 nM to 0.06 nM. The system looses bistability beyond a narrow range of PTP1B concentration between 0.02 nM to 0.05 nM. Thus, the response of GLUT4 translocation to insulin is particularly sensitive to PTP1B concentration. Influence of feedback on GLUT4 translocation The feedback effect of active Akt on PTP1B was studied by increasing the Akt concentration (Fig. 5A) and by changing the percentage feedback at a fixed Akt concentration (Fig. 5B). As shown in Fig. 5A, increase in Akt concentration amplifies the signal by maintaining bistable response. Similarly, by increasing the percentage feedback at a fixed Akt concentration, (Fig. 5B) the degree of bistability dramatically increased, while not influencing the threshold concentration required for switching-on the response. The bistable response was not observed when percentage feedback was smaller or in absence of feedback loops. In absence of receptor internalization, 65% inhibition of PTP1B by Akt was required to display a bistable response, whereas, inclusion of receptor internalization demonstrated bistability even at 25% inhibition of PTP1B. Figure 5 Influence of feedback effects on bistable insulin-stimulated GLUT4 translocation. A. Bistable response with increase in the concentration of Akt representing increased non-linearity due to zero order ultrasensitivity. Dose response curves obtained at different Akt concentrations: Curve 'a', 0.01 nM; Curve 'b', 0.03 nM; Curve 'c', 0.05 nM. B. Influence of percentage of feedback effects on dose response curve of insulin-stimulated GLUT4 translocation. The percentage feedback represents the percentage by which the dephosphorylation ability of PTP1B is impaired. Dose response curves obtained: Curve 'a' in absence of feedback; Curve 'b' 25% feedback effect; Curve 'c' 67% feedback effect; Curve 'd' 90% feedback effect. The steady state analysis of metabolic insulin-signaling pathway demonstrated signal amplification as signal propagates down the cascades. The amount of insulin required for 50% activation of insulin receptor, IRS-1, PIP3, Akt, PKC-ζ and GLUT4 was estimated to decrease in presence or absence of feedback loops (results not shown). Effect of system parameter values on GLUT4 translocation In addition to genetic variation at the protein expression levels in diseased conditions, mutational changes can also alter the system parameters and thereby modify the final output response. To examine the influence of system parameter values on insulin-stimulated GLUT4 translocation, we have analyzed the performance of insulin signaling pathway to variations in key parameter values such as, dissociation constant and Michaelis-Menten constant. Increase in dissociation constant quantifying the interaction between insulin-receptor and phosphorylated IRS-1-PI3K shows an increase in the half saturation constant indicating higher requirement of insulin over the physiological concentration (Fig. 6A and 6B). The system becomes monostable at very low values of dissociation constants. Similarly, decrease in the Michaelis-Menten constant of the dephosphorylation cycles, also increases the half saturation constant, thus decreasing the insulin sensitivity (Fig. 6C). Simulation results indicate that, the alterations in binding constant of allosteric interactions and Michaelis-Menten constants in modification-demodification cycles in the insulin-signaling pathway can result in insulin resistance or diabetes. Figure 6 Effect of key system parameter values on GLUT4 translocation. Curve 'a' shows half maximal concentration of insulin required to switch-on GLUT4 translocation. Curve 'b' shows half maximal concentration of insulin required to switch-off GLUT4 translocation. Arrow indicates parameter used in the simulation. A. Half maximal concentration of insulin required for GLUT4 translocation at different values of dissociation constant [Kd2] for binding of second molecule of insulin to phosphorylated insulin bound receptor. Simulated results indicate decreased insulin sensitivity when Kd2 increased. B. Half maximal concentration of insulin required for GLUT4 translocation at different values of dissociation constant [Kd3] for binding of phosphorylated IRS-1 to PI3K species. Simulated results indicate decreased insulin sensitivity when Kd3 increased. C. Half maximal concentration of insulin required for GLUT4 translocation at different values of Michaelis-Menten constant [Km2] for dephosphorylation of phosphorylated IRS1 by PTP1B. Simulated results indicate decreased insulin sensitivity when Michaelis-Menten constant [Km2] was decreased due to increased affinity with dephosphorylating enzyme. Discussion In this work we have demonstrated that, the dose response curves of fractional GLUT4 concentration on plasma membrane at various concentration of insulin exhibit hysteresis-a property of bistable systems. The analysis of bistable response in presence of feedback loops was done at varying concentration of signaling components and system parameters in physiological range. The overall response of insulin demonstrated signal amplification as the signal propagates down the cascade, thus requiring less insulin for GLUT4 translocation. The insulin sensitivity increased by increasing the concentration of proteins that amplify the insulin action and decreasing the concentration of proteins that attenuate insulin-signaling pathway. This indicates that the bistability and the half saturation constant are dependent on the component concentrations and system parameters. It is known that defects in insulin signaling pathway leads to pathological conditions like diabetes, wherein normal or elevated levels of insulin produces impaired biological response. This characteristic decrease or increase in insulin sensitivity is mainly attributed to post-receptor defects including mutational changes in protein expression levels or other parameters like dissociation constants and Michaelis-Menten constants [13,33]. Numerous experimental studies like targeted deletions/mutations of signaling components have yielded insights about the disease states. In the present work, to study the influence of pathological conditions on final output response of insulin, the concentration of individual signaling components was varied over a wide range, by keeping other parameters constant. The predicted results are consistent with various reported experimental observations and thus validate our steady state model. (i) Decreased concentration of phosphorylated insulin receptor and IRS-1 are observed in muscle from morbidly obese subjects [34] and those with diabetes [35]. This decreased phosphorylation can be either due to decrease in protein concentration itself or impaired phosphorylation event. (ii) Increase in the level and activity of several tyrosine phosphatases (PTP1B) was found to be associated with insulin resistance and reduced insulin sensitivity [12,13,33,36]. (iii) Overexpression of PI3K and its downstream targets Akt and PKC in cell culture models increased GLUT4 translocation [12]. (iv) Decrease in the association of PI3K with phosphorylated IRS-1 and subsequent activation appears to be a characteristic abnormality in type 2 diabetes and obesity [13,33-35]. (v) Single gene knockout experiments have shown that, mice with PTP1B knockout [37], mice with SHIP2 knockout [38] and targeted deletion of PTEN in murine lever [39], all results in hypersensitivity towards insulin. In the present work, though we have not done in-silico perturbation analysis by deleting a particular protein, we have changed the concentration of specific protein over wide range to bring about the similar effect of deficiency. Thus, our simulation results show that the insulin sensitivity dramatically increased when we decreased the concentration of phosphatases like PTP1B, PTEN and SHIP2. Increase in the concentration of Akt, makes the signal amplification increased along with slight increase in the degree of bistability. This effect is brought about by the enhanced nonlinearity in the feedback loop due to zero order ultrasensitivity [28] imposed by increasing the concentration of Akt or percentage feedback. At high Akt concentration (or when overexpressed), the system can respond in constitutively active mode or might also function as a memory module. That is, once insulin switches on the system, the high Akt concentration or percentage feedback by itself can keep the switch on even after insulin is removed. This may be the reason for the experimental observation of insulin independent GLUT4 translocation to plasma membrane when Akt is overexpressed [12,40]. This insulin independent translocation of GLUT4 is thought to be due to activation of PI3K independent pathway or by amplification of residual signal. Our analysis indicates that the inherent feedback structure present in the insulin-signaling pathway by itself can induce this phenomenon. Does GLUT4 translocation show a bistable response to insulin in-vivo? Bistability has been shown to be the outcome of regulatory structure with feedback loops and non-linearity in the system [41]. The non-linearity in the system is brought about by an ultrasensitive response typically obtained through enzyme cascades. Such ultrasensitive responses exhibit steep dose response curves with Hill coefficient greater than one [1]. The cascade systems use energy for their operation and are optimally operated under zero order sensitivity (i.e., cascades operating under saturating interconvertable enzymes) requiring minimum energy [42,43]. Presence of feedback loops may further optimize the requirement of energy. Enzyme cascades and multiple positive feedback loops are observed in insulin-signaling pathway. Experimental results have shown that the dose response curve of insulin-stimulated glucose uptake is subsensitive with a Hill coefficient of about 0.8 [calculated from ref. [32]]. Thus the question arises as to what may be the significance of the cascade signaling system with positive feedback loops in insulin signaling pathway. The reason for this discrepancy may be because, the experimental data represents a profile of glucose uptake in ensemble of cells [32], and furthermore, glucose uptake may not be proportional to the amount of GLUT4 on cell surface [18]. Recently, bistability has been experimentally demonstrated in many cellular regulation systems [10]. Experiments on cellular differentiation and cell-cycle progression have shown that, to demonstrate bistability it is essential to measure the amount of input required to switch-on the system from a low activity state to a high activity state, and amount of input required to hold-on the system in high activity state [3-5]. Reynolds et al. [8], have shown experimentally that, the reaction network of PTPase inhibition by activated epidermal growth factor receptor (EGFR, a receptor tyrosine kinase, RTK) through reactive oxygen species, can generate highly amplified and switch like bistable response to a threshold concentration of ligand stimulus. In this system, PTPase is a negative regulator of active RTK and thus, PTPase inhibition by reactive oxygen species serves as a positive feedback loop. Our simulation results indicate that similar bistable response can be obtained in insulin-stimulated GLUT4 translocation because of the positive feedback loops (inhibitory action of Akt on PTP1B). Though experimental verification of this property is awaited, there are indications that insulin signaling pathway possesses many requisite components to exhibit bistable response. The simulation results presented here showed that, the ultrasensitivity in absence of feedback effects and the regulatory structure of feedback loops are main reasons for a bistable response. Other than the positive feedback loops considered in the present work, Insulin signaling system is also known to contain many feedback loops which have not been entirely characterized [15]. One possible reason for having so many interlocking positive feedback and negative feedback loops may be to cause oscillations in GLUT4 translocation or to make the bistability of GLUT4 translocation – more robust. Recently, it has been shown that insulin stimulation in a variety of cell types elicit a rapid production of hydrogen peroxide and which reversibly inhibit PTP1B enhancing propagation of the early insulin signal [44]. This regulatory mechanism was also found to be essential for PI3K mediated Akt activation, GLUT4 translocation to plasma membrane and insulin-stimulated glucose uptake [45]. However, unlike EGFR system [8] existence of bistable behavior is yet to be shown in insulin signaling system. In insulin signaling pathway other than GLUT4, proteins like Akt and PKC get translocated to plasma membrane and such inter-compartmental translocations can also exhibit switch like bistable response [9]. It is becoming clear that quantification studies have to be performed in single cell rather than cell populations [19]. This is true especially while addressing the system level questions like ultrasensitivity, bistability and oscillations [4-7,46]. Recently, this was also found to be of immense value in case of insulin signaling pathway to uncover the PIP3 activation mode [47]. Simultaneous measurement of PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes suggested that a threshold for PIP3 signals exists. Below this threshold, GLUT4 transporters are minimally inserted into the plasma membrane and their insertion increases once this threshold is overcome. In summary, it is essential to show through experiments that, the stimulus-response curve for insulin-stimulated GLUT4 translocation exhibits hysteresis, – a distinctive splitting in stimulus response curve. There should be a particular concentration of insulin, which is not sufficient to translocate GLUT4 to plasma membrane, but is sufficient to maintain GLUT4 on plasma membrane. What may be the significance of such a bistable behavior in GLUT4 translocation? Though there is no obvious answer to this question yet, it is known that, bistability can maintain a biological response even when the input stimulus is brief and the high activity level is maintained only as long as the system requires. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-receptor complex. This internalization of receptor has been implicated in receptor down regulation, attenuation of insulin sensitivity and insulin clearance from the circulation [12,13]. Thus a brief input stimulation should be sufficient to balance the translocation of GLUT4 to plasma membrane and its reversal depending on glucose concentration. Additionally, due to small absolute stimulus concentrations, the probability of noise occurrence is high. A bistable response having distinct threshold concentration to switch-on and switch-off offers advantage in handling noise. In cellular regulation, different structural motifs such as enzyme cascades, feedforward control and multiple feedback loops yield complex regulatory networks. These are shown to be responsible for exhibiting system level properties including bistability and robustness [10,48,49]. Other than multiple feedback loops, structural regulatory motifs like multisite covalent modification cycles have been shown to induce bistability [50]. The interconnections between these regulatory motifs at the system level may elicit a multistable response to particular stimuli, which have to be theoretically uncovered and demonstrated through experiments. Abbreviations used GLUT4: Glucose-transporter isoform 4; IRS: Insulin-receptor substrate; PI3K: Phosphatidylinositol-3-kinase; PIP3: Phosphatidylinositol (PI)-3,4,5-tiphosphate (PI-3,4,5-P3); Akt: Protein kinase Akt or protein kinase B (PKB); PKC: Protein kinase C; PTP1B: Protein tyrosine phosphatase 1B; PTEN: 3' lipid phosphatase; SHIP2: 5' lipid phosphatase; Competing Interests None declared. Author's Contributions LG, VKM and KVV conceived and designed the experiments. LG performed the experiments. LG, VKM and KVV analyzed the data. VKM and KVV conceptualize the manuscript. All authors have read and approved the final manuscript Appendix Equilibrium relationships, rate equations, mass balance equations and model parameters used for simulation of metabolic insulin signaling system at steady state (refer Fig. 2 for nomenclature and interaction details). Equations were solved simultaneously, for evaluating fractional GLUT4 translocation at a particular insulin concentration, using fsolve function of Matlab (The MathWorks Inc. USA). Most of the values of model parameters for steady state analysis are taken from Sedaghat et al. [26]. Nomenclature, parameter values are: Component concentrations It, total insulin concentration varied over a wide range; Xt, total receptor = 0.003 nM; At, total IRS-1= 0.01 nM, Bt, total PI3-Kinase = 0.5 nM, PTENt, total PTEN= 0.007 nM; CP2t, total lipid = 0.01 nM; SHIP2t, total SHIP2 = 0.001 nM; Ft, total Akt+PKC-ξ = 0.02 nM, PTPmax, total PTP1B= 0.039 nM; Gt, total GLUT4 = 9 nM; E6t, total E6 = 0.001 nM; E8t, total E8 = 0.001 nM; Rate constants k0 = 2500 min-1; k = 0.2 min-1; k1= 4.16 min-1; k2 = 1.4 min-1; k3 = 50 min-1 (assumed); k4 = 42.1 min-1; k5 = 2.8 min-1; k6 = 3 min-1; k7 = 20 min-1 (assumed); k8 = 6.9 min-1; k9 = 0.11 min-1; k10 = 0.0012 min-1; k11 = 3.47 min-1 (assumed); k12 = 6.96*10-3 min-1; k13 = 0.17 min-1; kp = 0.461 min-1 ; kd = 1.67 × 10-18 min-1 ; ks = 1.67*10-25 nM min -1; Dissociation constants Kd1= 3.33 nM; Kd2 = 333.3 nM; Kd3 = 0.014 nM; Distribution coefficients Kd4 = 9 nM; Kd5 = 0.1 nM; Michaelis-Menten constants Kmr, Km1 to Km8 = 10-4 nM The total molar balances for different species are given below. It = I + XI + XIP + 2XI2P + PTP.XIP + A.XI2P + XIPi + 2XI2Pi + PTP.XIPi + PTP.XI2Pi     [A1] Xt = X + Xi + XI + XIP + XI2P + XIPi + XI2Pi + PTP.XIP + A.XI2P + PTP.XIPi + PTP.XI2Pi     [A2] At = A + AP + APB + A.XI2P + PTP.AP + APB.CP2     [A3] CP2t = CP2 + CP3 + CP2' + APB.CP2 + PTEN.CP3 + SHIP2.CP3 + SHIP2.CP3 + E6.CP2' + F.CP3     [A4] Ft = F + FP +CP3F + E8.FP     [A5] SHIP2t = SHIP2 + SHIP2.CP3     [A6] PTENt = PTEN + PTEN.CP3     [A7] PTPt = PTP + PTP.XIP + PTP.AP + PTP.XIPi + PTP.XI2Pi     [A8] Bt = B + APB + CP2.APB     [A9] E6t = E6 + E6.CP2'     [A10] E8t = E8 + E8.FP     [A11] Gt = GM + GC     [A12] Equilibrium relationships Rate expression with pseudo-steady state representation of complexes for modification-demodification cycles Receptor autophosphorylation and dephosphorylation cycle IRS-1 phosphorylation and dephosphorylation cycle Phosphorylation and Dephosphorylation of PI-4,5-P2, PI-3,4-P2 and PIP3 Phosphorylation and Dephosphorylation of Akt + PKC ==== Refs Ferrell JE Jr Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs Trends Biochem Sci 1996 21 460 466 9009826 10.1016/S0968-0004(96)20026-X Bagowski CP Ferrell JE Jr Bistability in the JNK cascade Curr Biol 2001 11 1176 1182 11516948 10.1016/S0960-9822(01)00330-X Becskei A Seraphin B Serrano L Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion EMBO J 2001 20 2528 2535 11350942 10.1093/emboj/20.10.2528 Pomerening JR Sontag ED Ferrell JE Jr Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2 Nat Cell Biol 2003 5 346 351 12629549 10.1038/ncb954 Sha W Moore J Chen K Lassaletta AD Yi CS Tyson JJ Sible JC Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts Proc Natl Acad Sci USA 2003 100 975 980 12509509 10.1073/pnas.0235349100 Xiong W Ferrell JE Jr A positive-feedback-based bistable memory module that governs a cell fate decision Nature 2003 426 460 465 14647386 10.1038/nature02089 Ozbudak EM Thattai M Lim HN Shraiman BI Oudenaarden AV Multistability in the lactose utilization network of Escherichia coli Nature 2004 427 737 740 14973486 10.1038/nature02298 Reynolds AR Tischer C Verveer PJ Rocks O Bastiens PIH EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation Nature Cell Biol 2003 5 447 453 12717446 10.1038/ncb981 Teruel MN Meyer T Parallel signal-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module Science 2002 295 1910 1912 11884760 10.1126/science.1065028 Ferrell Jr JE Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability Curr Opin Chem Biol 2002 14 140 148 10.1016/S0955-0674(02)00314-9 Saltiel AR Kahn CR Insulin signaling and the regulation of glucose and lipid metabolism Nature 2001 414 799 806 11742412 10.1038/414799a Nystrom FH Quon MJ Insulin signalling: Metabolic pathways and mechanisms for specificity Cell Signal 1999 11 563 574 10433517 10.1016/S0898-6568(99)00025-X Shepherd PR Kahn BB Glucose transporters and insulin action: implication for insulin resistance and diabetes mellitus N Engl J Med 1999 341 248 257 10413738 10.1056/NEJM199907223410406 Bryant NJ Govers R James DE Regulated transport of the glucose transporter GLUT4 Nat Rev Mol Cell Biol 2002 3 267 277 11994746 10.1038/nrm782 Johnston AM Pirola L Obberghen EV Molecular mechanisms of insulin receptor substrate protein-mediated modulation of insulin signaling FEBS Lett 2003 546 32 36 12829233 10.1016/S0014-5793(03)00438-1 Saltiel AR Pessin JE Insulin signaling pathways in time and space Trends Cell Biol 2002 12 65 71 11849969 10.1016/S0962-8924(01)02207-3 Watson RT Kanzaki M Pessin JE Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes Endocrine Rev 2004 25 177 204 15082519 10.1210/er.2003-0011 Kandror KV A long search for GLUT4 activation Sci STKE 2003 PE5 12582199 Meyer T Teruel MN Fluorescence imaging of signaling networks Trends Cell Biol 2003 13 101 106 12559761 10.1016/S0962-8924(02)00040-5 Shymko RM Dumont E De Meyts P Dumont JE Timing-dependence of insulin-receptor mitogenic versus metabolic signalling: a plausible model based on coincidence of hormone and effector binding Biochem J 1999 339 675 683 10215607 10.1042/0264-6021:3390675 Wanant S Quon MJ Insulin receptor binding kinetics: modeling and simulation studies J Theor Biol 2000 205 355 364 10882558 10.1006/jtbi.2000.2069 Quon MJ Campfield LA A mathematical model and computer simulation study of insulin receptor regulation J Theor Biol 1991 150 59 72 1890848 Quon MJ Advances in kinetic analysis of insulin-stimulated GLUT-4 translocation in adipose cells Am J Physiol 1994 266 E144 E150 8304439 Holman GD Lo Leggio L Cushman SW Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools J Biol Chem 1994 269 17516 17524 8021259 Quon MJ Campfield LA A mathematical model and computer simulation study of insulin-sensitive glucose transporter regulation J Theor Biol 1991 150 93 107 1890850 Sedaghat AR Sherman A Quon MJ A mathematical model of metabolic insulin signaling pathways Am J Physiol Endocrinol Metab 2002 283 E1084 E1101 12376338 Elmendorf JS Signals that regulate GLUT4 translocation J Membr Biol 2002 190 167 174 12533782 10.1007/s00232-002-1035-3 Goldbeter A Koshland DE Jr An amplified sensitivity arising from covalent modification in biological systems Proc Natl Acad Sci USA 1981 78 6840 6844 6947258 Di Guglielmo GM Drake PG Baass PC Authier F Posner BI Bergeron JJM Insulin receptor internalization and signaling Mol Cell Biochem 1998 182 59 63 9609114 10.1023/A:1006883311233 Shi K Egawa K Maegawa H Nakamura T Ugi S Nishio Y Kashiwagi A Protein-Tyrosine Phosphatase 1B Associates with Insulin Receptor and Negatively Regulates Insulin Signaling without Receptor Internalization J Biochem 2004 136 89 96 15269244 10.1093/jb/mvh094 Ravichandran LV Chen H Li Y Quon MJ Phosphorylation of PTP1B at Ser (50) by Akt impairs its ability to dephosphorylate the insulin receptor Mol Endocrinol 2001 15 1768 1780 11579209 10.1210/me.15.10.1768 Stagsted J Hansen T Roth RA Goldstein A Olsson L Correlation between insulin receptor occupancy and tyrosine kinase activity at low insulin concentrations and effect of major histocompatibility complex class I-derived peptide J Pharmacol Exp Ther 1993 267 997 1001 8246175 Pessin JE Saltiel AR Signaling pathways in insulin action: molecular targets of insulin resistance J Clin Invest 2000 106 165 169 10903329 Goodyear LJ Giorgino F Sherman LA Carey J Smith RJ Dohm GL Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects J Clin Invest 1995 95 2195 2204 7537758 Bjornholm M Kawano Y Lehtihet M Zierath JR Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation Diabetes 1997 46 524 527 9032113 Ahmad F Azevedo JL Cortright R Dohm GL Goldstein BJ Alterations in skeletal muscle protein-tyrosine phosphatase activity and expression in insulin-resistant human obesity and diabetes J Clin Invest 1997 100 449 458 9218523 Elchebly M Payette P Michaliszyn E Cromlish W Collins S Loy AL Normandin D Cheng A Himms-Hagen J Chan CC Ramachandran C Gresser MJ Tremblay ML Kennedy BP Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene Science 1999 283 1544 1548 10066179 10.1126/science.283.5407.1544 Clément S Krause U Desmedt F Tanti JF Behrends J Pesesse X Sasaki T Penninger J Doherty M Malaisse W Dumont JE Le Marchand-Brustel Y Erneux C Hue L Schurmans S The lipid phosphatase SHIP2 controls insulin sensitivity Nature 2001 409 92 7 11343120 10.1038/35051094 Stiles B Wang Y Stahl A Bassilian A Lee WP Kim YJ Sherwin R Devaskar S Lesche R Magnuson MA Wu H Liver-specific deletion of negative regulator PTEN results in fatty liver and insulin hypersensitivity Proc Natl Acad Sci USA 2004 101 2082 2087 14769918 10.1073/pnas.0308617100 Cong LN Chen H Li Y Zhou L McGibbon MA Taylor SI Quon MJ Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells Mol Endocrinol 1997 11 1881 1890 9415393 10.1210/me.11.13.1881 Angeli D Ferrell JE JrSontag ED Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems Proc Natl Acad Sci USA 2004 101 1822 1827 14766974 10.1073/pnas.0308265100 Shacter E Chock PB Stadtman ER Energy consumption in a cyclic phosphorylation/dephosphorylation cascade J Biol Chem 1984 259 12260 12264 6090463 Goldbeter A Koshland DE Jr Energy expenditure in the control of biochemical systems by covalent modification J Biol Chem 1987 262 4460 4471 3558349 Mahadev K Zilbering A Zhu L Goldstein BJ Insulin-stimulated hydrogen peroxide reversibly inhibits protein tyrosine phosphatase 1B in-vivo and enhances the early insulin action cascade J Biol Chem 2001 276 21938 21942 11297536 10.1074/jbc.C100109200 Mahadev K Wu X Zilbering A Zhu L Todd J Lawrence R Goldstein BJ Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes J Biol Chem 2001 276 48662 48669 11598110 10.1074/jbc.M105061200 Ferrell JE JrMachleder EM The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes Science 1998 280 895 898 9572732 10.1126/science.280.5365.895 Tengholm A Meyer T A PI3-kinase code for insulin-triggered insertion of glucose transporters into the plasma membrane Curr Biol 2002 12 1871 1876 12419189 10.1016/S0960-9822(02)01223-X Bhalla US Ram PT Iyengar R MAP kinase phosphatase as a locus of flexibility in a mitogen-activated protein kinase signaling network Science 2002 297 1018 1023 12169734 10.1126/science.1068873 Venkatesh KV Bhartiya S Ruhela A Multiple feedback loops are key to dynamic performance of tryptophan regulation in Escherichia coli FEBS Lett 2004 563 234 240 15063755 10.1016/S0014-5793(04)00310-2 Markevich NI Hoek JB Kholodenko BN Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades J Cell Biol 2004 164 353 359 14744999 10.1083/jcb.200308060

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==== Front Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-1-31530788910.1186/1742-4682-1-3ResearchRegulatory role of E-NTPase/E-NTPDase in Ca2+/Mg2+ transport via gated channel Schreiber Hans M [email protected] Subburaj [email protected] Division of Gastroenterology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA2 Departments of Microbiology and Immunology, School of Medicine, PO Box 25056, University of Texas Medical Branch, 300 University Boulevard, Galveston, Texas, 77550 USA2004 12 8 2004 1 3 3 31 5 2004 12 8 2004 Copyright © 2004 Schreiber and Kannan; licensee BioMed Central Ltd.2004Schreiber and Kannan; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background E-NTPase/E-NTPDase is activated by millimolar concentrations of Ca2+ or Mg2+ with a pH optimum of 7.5 for the hydrolysis of extracellular NTP and NDP. It has been generally accepted that E-NTPase/E-NTPDase plays regulatory role in purinergic signalling, but other functions may yet be discovered. Results In this article it is proposed on the basis of published data that E-NTPase/E-NTPDase could play a role in the influx and efflux of Ca2+and Mg2+ in vivo. Conclusions Attenuation of extracellular Ca2+ influx by rat cardiac sarcoplasmic anti-E-NTPase antibodies and oligomerization studies on mammalian CD39 conclusively point towards the existence of a new channel in the membrane. Further studies on these properties of the E-NTPase/E-NTPDase may provide detailed mechanisms and identify the potential patho-physiological significance. ==== Body Background The mechanism by which [Ca2+]i is increased in excitable cells differs from that obtaining in non-excitable cells. Excitable cells exhibit an action potential, a substantial general depolarization of the plasma membrane, in response to depolarizing stimuli; influx of Ca2+ occurs via plasma membrane Ca2+ channels and/or release from sarco (endo) plasmic reticulum via ryanodine-receptor Ca2+ channels which regulate the excitation – contraction coupling [1,2]. The factors that determine the extent of Ca2+ entry are (i) magnitude of the membrane potential and (ii) magnitude of the transmembrane Ca2+ gradient. These two factors also determine whether Ca2+ or Mg2+ enters and the time (probably milliseconds) that elapses between channel opening and termination of Ca2+ or Mg2+ transport [3]. In non-excitable cells, the increase in [Ca2+]i results from influx of Ca2+ across the plasma membrane and Ca2+ release from the endoplasmic reticulum. Ca2+ release from the SER depends on the binding of inositol 1,4,5-triphosphate (InsP3) to its receptor Ca2+channels, and also on Ca2+ binding to ryanodine receptor – Ca2+channels. Ca2+ is removed from the cell by the following means. i: the sarco (endo) plasmic reticular Ca2+ pump ATPase (SERCA), which transports Ca2+ from the cytoplasm into the SER lumen (~70% of the activator Ca2+); ii: The plasma membrane Ca2+ pump ATPase (PMCA), which exports Ca2+ across the plasma membrane (~1% of the activator Ca2+); iii: Mitochondrial Ca2+Uniporters (mCa2+ uniporters), which transport Ca2+ into mitochondria (~1% of the activator Ca2+);iv: the Na+/Ca2+ exchanger (28% of the activator Ca2+). This last transport system is reversible but under normal physiological conditions, in the Ca2+ extrusion mode, it exhibits a stoichiometry of 3 Na+influx/1 Ca2+ efflux [4]. Ca2+ enters animal cells via (i) voltage-operated Ca2+channels (VOCC), (ii) ligand gated non-specific cation channels (LGCCS), and (iii) stretch/receptor activated non-specific Ca2+ channels (RACC) [4,5]. A "receptor operated Ca2+ channel" (ROCC) is defined as a plasma membrane Ca2+ channel other than VOCC or RACC. VOCC opening depends on membrane depolarization, whereas RACC opening depends on both direct and indirect activation of membrane bound receptors. In contrast, ROCC opening depends solely on agonist-receptor interaction. It has also been suggested that mobile intracellular messengers such as elevated [Ca2+]i play a role in ROCC opening [5,6]. Different types of ROCC are activated (opened) by diverse cell signaling mechanisms such as ligand specificity, increase in [Ca2+]I, increase in [cAMP]i [7] and activation/inactivation of specific trimeric G proteins [8]. Opening of Ca2+ channels must be a highly regulated event involving physical movement of channel components inclusive of the alteration in channel protein conformation; Also, an extracellular source of free energy (ΔG) could be of critical importance. This might be supplied by E-NTPase/E-NTPDase mediated hydrolysis of NTP/NDP. Co-ordination of this process might play a role in the opening of Ca2+ channels, independently of membrane depolarization or other factors. The biochemical, structural, and functional properties of E-type nucleotidases have been covered in several excellent reviews: i. Extracellular metabolism [9]; ii. purine signalling [10,11]; iii. adhesion [12]; iv. transporter functions [13]; v. pathophysiology [14,15]. Rationale for the proposed hypothesis: E-NTPase/E-NTPDase mediated Ca2+/Mg2+ transport It has been suggested that Ca2+ entry during the slow inward current in normal myocardium involves membrane-bound channels potentially controlled and/or regulated by metabolic energy transfer from unknown sources, though Ca2+ enters the cell down its concentration gradient [16]. Electrical stimulation and membrane phosphorylation by cAMP-dependent protein kinase have been shown to increase E-NTPase/E-NTPDase activity. Metal ions such as Mn2+, Co2+, Ni2+ and La2+ that attenuate Ca2+ influx also inhibit the E-NTPase. In the late stages of heart failure the E-NTPase is down regulated. Activation of E-NTPase by various concentrations of Ca2+ has been shown to correlate linearly with cardiac contractile force development [17]. "Calcium paradox" is defined as irreversible functional and structural protein loss in the isolated heart that is first perfused with Ca2+-free buffer and then reperfused with Ca2+-containing buffer [18]. E-NTPase activity is highest during the initial phases of reperfusion, which might favour the initial Ca2+ influx that causes Ca2+ overload. During the later stages of reperfusion with Ca2+-containing buffer there is a loss of E-NTPase activity. During mild stages of Ca2+ paradox, E-NTPase retains its function and continues to favour Ca2+ influx, resulting in the development of intracellular Ca2+ overloads. However, during severe stages of calcium paradox, impaired E-NTPase activity may contribute to irreversible failure of contractile force recovery [19]. To date there is no report describing the detailed mechanism of E-NTPase/E-NTPDase-mediated channel gating and its role in Ca2+/Mg2+ transport. In this article an attempt is made to delineate the molecular mechanism of Ca2+/Mg2+ transport, identifying the source of energy and the activation and termination of the process. The central issues are: a. How the metabolic energy from nucleotide hydrolysis is effectively utilized in channel opening; b. What stage of the opening/closing cycle requires energy; c. By what (probable) mechanism the proposed scheme is completed; d. How, if at all, homeostasis is affected The current hypothetical proposal is set out in three sections with appropriate illustrations. Phase I: Activation identifies the evidence that leads to the current proposal and describes how the metabolic energy from nucleotide triphosphate hydrolysis is utilised to assemble a functional homo-oligomer of the E-NTPase/E-NTPDase, forming a channel that is subsequently opened. Phase II: Suggested: Ca2+/Mg2+Transport Describes, with supporting evidence, how the energy released from [NTP] o/ [NDP] o hydrolysis might be utilized for opening the channel formed by the homo-oligomeric ENTPase/E-NTPDase. Phase III: Termination of the transport processes outlines the intracellular and extracellular factors that would influence the termination of the Ca2+/Mg2+ transport processes, and the experimental evidence obtained in favor of the whole proposal. Phase I: Activation of E-NTPase/E-NTPDase and channel formation Membrane depolarization could locally alter protein conformation. This in turn could potentially induce post-translational modification in the (intracellular) monomer subunits of the E-NTPase/E-NTPDase, followed by translocation to the membrane (depending on the tissue type(s) and functional requirement(s)) (Fig. 1). Fig. 2 shows the proposed functional state of the E-NTPase/E-NTPDase after oligomerization and assembly in the membrane to form a gated Ca2+/Mg2+ channel. Fig. 3, indicates that the oligomerized E-NTPase/E-NTPDase is likely to possess sensors to control the opening and closing of the Ca2+/Mg2+ channel gate. Fig. 4, represents an interior view of the E-NTPase/E-NTPDase in the functional state after oligomerization and assembly in the membrane. Figure 1 Phase I: Activation. Based on direct experimental evidence, suppose that in response to electrical stimuli, an increased phosphatidylinositol turnover leads to elevated intracellular phospholipid. This in turn could induce post-translational modification of the monomer subunits of E-NTPase/E-NTPDase in the intracellular milieu. Subsequently, the monomers are translocated to the membrane, depending on the tissue type(s) and functional requirement(s). Figure 2 Phase I: Activation. Proposed model for E-NTPase/E-NTPDase in a functional state after oligomerization and assembly in the membrane, functioning as a gated channel. Figure 3 Phase I: Activation. The oligomerized E-NTPase/E-NTPDase would probably possess hypothetical sensors acting to open/close the gates. Figure 4 Phase I: Activation. Interior view of E-NTPase/E-NTPDase in a functional state in the membrane. Probable energy sources and other significant factors are as follows. The source of extracellular nucleotides could be spontaneous release from dead cells or exocytosis from live/damaged cells [20]. In ocular ciliary epithelial cells, ATP is released in hypotonic conditions, and this release is inhibited by NPPB (5-nitro-2-(3-phenyl propylamine benzoic acid), a potent inhibitor of CFTR (cystic fibrosis transmembrane receptor) and p-glycoprotein mediated ATP release [21]. On the other hand, the endogenous CD39 of oocytes transforms under hypertonic conditions to a conformation mediating ATP transport to the extracellular environment, either by exocytosis or by acting as an ion channel [22,23]. However, under what conditions (hyper-or hypotonic) might CD39 assume an extracellular nucleotide hydrolyzing activity; and under those conditions, can this property be coupled to ion influx? This question remains unanswered. At normal physiological temperature in presence of divalent succinyl CoA, Con A mediates the oligomerization of E-NTPase monomers/dimers to form a holoenzyme with enhanced activity. Eosin iodoacetamide (EIAA), a fluorescein iodoacetamide that forms thioester bonds with cysteine at neutral pH, enhances chicken gizzard ecto-ATPase activity [24]. There are ten conserved cysteine residues in E-NTPase (with additional cysteine residues in the N-terminal region that are known to mediate disulfide bond formation, essential in oligomerization). CD39, an ecto-Ca2+/Mg2+ apyrase that hydrolyses ATP and ADP [25], forms tetramers and might act as a bivalent cation channel. However, the precise mechanism and functional properties are not known at present. CD39 expression is associated with ATP release; it was speculated that ATP release (along with drugs) into the extracellular milieu is followed by the hydrolysis of the extracellular nucleotides by CD39 [26]. Furthermore, native CD39 (ecto-ATP/Dase/ apyrase) forms tetramers upon oligomerization. Loss of either of the two transmembrane domains of rat CD39 ecto-ATP/Dase impairs enzyme activity. It has been suggested that the functional (holoenzyme) E-NTPase/E-NTPDase is a homotrimer in mammals. Differences in enzyme activity among different species have been attributed to variations in the interaction among the monomers resulting in homotrimeric holoenzyme formation (66 kDa-ATPase) [27]. It seems clear that changes in the conformation of the E-NTPase/E-NTPDase could mediate changes in the channel transport function. Phase II: Ca2+/Mg2+ Transport Fig. 5a, illustrates the possible utilization of the energy released from [NTP] o /[NDP] o hydrolysis (-7.3 kcal mol-1 or by formation of AMP, -10.9 kcal/mol-1) for opening the channel formed by the homo-oligomeric E-NTPase/E-NTPDase. This channel is postulated to open and close in response to energy availability (Fig. 5b). Fig. 6A, is an artist's impression of the three-dimensional configuration of the E-NTPase/E-NTPDase in vivo. Ca2+ might enter the cell and excess Mg2+ might leave by the influx and efflux mechanisms depicted in Fig 6b. Figure 5 Phase II: Ca2+/Mg2+ Transport. (A) Free energy released from ATP hydrolysis by E-NTPase on the outer membrane surface would yield -7.3 kcal mol-1 or by formation of AMP by E-NTPDase would yield -10.9 kcal mol-1. (B) The energy is utilized for opening the channel formed by the E-NTPase/E-NTPDase, by altering the conformation of the sensors. This altered conformation has an inherent channel-opening effect; loss of the energy source causes the sensors to revert to the resting state, which corresponds to channel closing. Figure 6 Phase II: Ca2+/Mg2+ Transport. (A) Three-dimensional impression of the E-NTPase/E-NTPDase in vivo. (B) It is possible that Ca2+ can enter the cell and excess Mg2+ can leave via the influx/efflux mechanisms depicted in the figure. The opening of the slow inward Ca2+ current channel in cardiac sarcolemma during the plateau phase of the action potential requires ATP [28]. Furthermore, protein kinase-A (PKA) dependent phosphorylation appears to mediate the increase in Ca2+ influx in hormonal modulation of that process [29]. A similar model has been proposed for sodium channels in nerve membranes, in which a cycle of phosphorylation and dephosphorylation is proposed for opening and closing [30]. Other corroborating evidence implicating E-NTPase in Ca2+/Mg2+ transport via the gated channel is briefly summarised. Rat cardiac sarcolemmal E-NTPase has considerable sequence homology with the human platelet thrombospondin receptor CD36 [31]. An antibody directed against the purified E-NTPase blocked the increase in intracellular calcium concentration, implying that the E-NTPase plays an unknown but significant role in the delayed Ca2+ influx or Mg2+ efflux during the plateau phase of the action potential (Unpublished observation). Activation of E-NTPase by millimolar concentrations of Ca2+ and electrical stimulation is linearly related to the contractile force developed in the myocardium [32]. Gramicidin S inhibits the E-NTPase activity and it attenuates the slow channel efflux in perfused frog left ventricles. Based on these observations, we propose that E-NTPase might be involved in providing energy for Ca2+/Mg2+ influx-efflux in the cardiac sarcolemma, opening the channel formed by the E-NTPase/E-NTPDase protein by altering the conformation of the sensors. The altered channel sensor conformation opens the channel; loss of the energy source allows the sensors to revert to the resting state, which corresponds to channel closing. There are at least two Mg2+ transport systems: (a) rapid transport down the concentration gradient and (b) efflux in low Ca2+ Ringer during ventricular perfusion in vitro. In rat liver mitochondria, 50 nM cAMP or 250 μM ADP induced rapid loss of 6 mmol of Mg2+/mg protein coupled with the stimulation of ATP efflux. This effect was specific and was blocked by adenosine nucleotide translocase inhibitors. Evidently cAMP acts as a mobilizer of Mg2+ in isolated rat liver mitochondria. Adenine nucleotide translocase is the cAMP target [33]. Myocardial Mg2+ content is maintained at physiological level by the sarcolemmal transport system, which pumps Mg2+ across the plasma membrane when the extracellular [Mg2+]o concentration is <1 mM and restores [Mg2+]i when the heart is perfused with Ringer buffer containing 5 × 10-7 M Mg2+. Failure of either of these two transport mechanisms may result in a rise in [Mg2+]i, impairing the contractile machinery of the myocardium [34]. Gramicidin S inhibits total Mg2+ efflux in the myocardium, while epinephrine restores Mg2+ efflux and contractile force development in the frog ventricle perfused with 10 mM Mg2+. It should be pointed out that both E-NTPase activity and myocardial contraction and relaxation are inhibited by gramicidin S [35]. In the light of the evidence surveyed here, there would appear to be a significant functional role for activated E-NTPase in Ca2+ influx and Mg2+ efflux (or vice versa) in the myocardium. Phase III: Termination of the transport process Fig. 7 summarizes the possible means by which the transport process is terminated. There are several potential contributing factors that can be grouped into two categories, extracelluar and intracellular. Additional experimental evidence is indicated. Based on the heterologous expression of ecto-apyrase in COS cells in the presence of tunicamycin, glycosylation might be required for homo-oligomerization and nuclotidase activity. Conversely, deglycosylation might impair the E-type nucleotidase activity by weakening the monomer-monomer interaction and altering the tertiary and quaternary structures, result in the loss of holoenzyme. Essentially, glycosylation and deglycosylation of the ecto apyrase (HB6) monomer and the consequences for homodimer formation have been regarded as an on-off switch for ecto nucleotidase activity [36]. Figure 7 Phase III: Termination of the transport processes. (A) Several factors might contribute to the termination of Ca2+/Mg2+ transport via channel gating by E-NTPase/E-NTPDase: extracelluar and Intracellular. Additional experimental evidence is mentioned. Decreased flow of Ca2+/Mg2+ due to closing of the channel gate. Fig. 8a is a three-dimensional impression of the ecto-ATPase in vivo at the termination of ion transport. Fig. 8b illustrates how biochemical modifications such as deglycosylation of the E-NTPase/E-NTPDase oligomers might cause dissociation of the homo-oligomers to individual monomers This is a potential mechanism for the disassembly of the functional channel and closure of Ca2+ influx and Mg2+ efflux. Also, an increase in membrane fluidity induced by cholesterol oxidation might cause defective association or disassociation due to weak interaction among the E-NTPase monomers, whereas increased membrane cholesterol might sustain higher E-NTPase activity. Oligomerization of E-NTPase and associated increase of activity could also be responsible for the rapid termination of the purinergic response mediated by extracellular ATP [37]. Figure 8 Phase III: Termination of the transport processes. (A) Three-dimensional impression of the E-NTPase/E-NTPDase in vivo when termination of the ion transport function commences. (B) Biochemical modifications of the E-NTPase/E-NTPDase oligomers such as deglycosylation would probably cause instability, leading to dissociation of the homo-oligomers. Disassembly of the functional molecule would ensue, closing the Ca2+ influx and Mg2+ efflux processes, as portrayed in the figure. The extracellular nucleotide mediated activation of channel gating could be terminated by ecto (extracellular)-adenylate kinase, which catalyzes trans-phosphorylase activity (ADP+ADP→ ATP+AMP). This enzyme has a higher affinity for extracellular nucleotides than the dephosphorylating enzyme (E-NTPase/E-NTPDase) or ecto-nucleotide pyrophosphatase/phospho-diesterase (ATP→ AMP +ppi) [38]. As the transport process winds down, ecto-adenylate kinase mediated ATP generation might maintain the extracellular nucleotide level. However, the precise biochemical kinetic process by which this process is completed remains to be elucidated [39]. Pathophysiological Significance of E-type nucleotidase mediated Ca2+/Mg2+ transport Impairment of E-Type nucleotidases during Ca2+ paradox in isolated rat heart model warrants investigation of the molecular mechanism(s) involved. Knowledge obtained from these studies will elucidate the observed protective effects of anti-rat cardiac Ca2+/Mg2+-ecto-ATPase antibodies in ischemia reperfusion induced damage, which is a corollary of organ transplantation. Furthermore, the antiproliferative effect(s) of these antibodies in left anterior descending coronary artery smooth muscle cell(s) emphasize the need to explore more fully the hypothesis proposed in this article. Authors' contributions HMS participated and provided the hypothetical scheme of the gating mechanism with appropriate literature. SK conceived and carried out experimental part of the investigation and formulating the hypotheses. Abbreviations E-NTPase = Ecto or Extracellular Nucleotide triphosphatase; E-NTPDase = Ecto or Extracellular Nucleotide triphosphate diphosphohydrolase; [Ca2+]i = Intracellular Ca2+; [NTP]0 = Extracellular Nucleotide triphosphate; [NDP]0 = Extracellular Nucleotide diphosphate; ROCC = Receptor Operated Ca2+ channel; SER = Sarco (Endo) plasmic reticulum; [cAMP]i = Cytoplasmic or intracellular cAMP; PMCA = Plasma membrane Ca2+pump ATPase.VOCC = voltage-operated Ca2+ channel; LGCCS = Ligand gated non-specific cation channels. RACC = Stretch/Receptor activated non-specific Ca2+channels. SUR = Sulfonylurea Receptor Proteins. CD36 = Thrombospondin receptor on platelets. CD39 = Ecto Ca2+/Mg2+apyrase. Acknowledgements Data were obtained during graduate (Ph.D.) work (1993–1998) supported by a graduate fellowship (to S.K) from St. Boniface General Hospital Research Foundation, Winnipeg, Canada. T.R. Smith, Medicinal Chemistry, University of Warwick, Leamington Spa, and Warwickshire, United Kingdom prepared most of the models. This manuscript was prepared during the tenure of a post-doctoral fellowship (September 2000 – January 2001) supported by grants DK-52216 and DK-44237 from the National Institutes of Health, Bethesda, MD, and USA. T.L. Kirley, A.F. Knowles, L. Plesner, A.R. Beaudoin, A.Z. Herzberg, M. Handa, K.A. Jacobson, A. Froese, P. Zahradka, L.J. Murphy, N.N. Tandon, N. Abumrad, A. Ibrahimi, R. Lipsky, D. Perlmutter, S.H. Lin, H. Zimmermann and N.N. Tandon are acknowledged for donating reagents, advises, and expert opinion on investigation during the period 1991–1998. Dr. Hans M. Schreiber has passed away during the preparation of this manuscript. The authors do not have any competing financial or intellectual properties interests with CIBA-GEIGY Canada or Novartis Pharmaceuticals Inc, Switzerland. Modified reagents are part of the impending U.S. or International patent application(s). ==== Refs Catterall WA Structure and function of voltage-gated ion channels Annu Rev Biochem 1995 64 493 531 7574491 10.1146/annurev.bi.64.070195.002425 Dunlap K Luebke JI Turner TJ Exocytotic Ca2+ channels in mammalian central neurons Trends Neurosci 1995 18 89 98 7537420 10.1016/0166-2236(95)93882-X Rutecki PA Neuronal excitability: voltage-dependent currents and synaptic transmission J Clin Neurophysiol 1992 9 195 211 1375602 Clapham DE Calcium signaling Cell 1995 80 259 268 7834745 10.1016/0092-8674(95)90408-5 Berridge MJ Elementary and global aspects of calcium signalling J Exp Biol 1997 200 315 319 9050239 Putney JW JrBird GS The inositol phosphate-calcium signaling system in nonexcitable cells Endocr Rev 1993 14 610 631 8262009 10.1210/er.14.5.610 Applegate TL Karjalainen A Bygrave FL Rapid Ca2+ influx induced by the action of dibutylhydroquinone and glucagon in the perfused rat liver Biochem J 1997 323 463 467 9163339 Macrez-Lepretre N Kalkbrenner F Schultz G Mironneau J Distinct functions of Gq and G11 proteins in coupling alpha1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes J Biol Chem 1997 272 5261 5268 9030598 10.1074/jbc.272.8.5261 Zimmermann H Nucleotides and cd39: principal modulatory players in hemostasis and thrombosis Nat Med 1999 5 987 988 10470068 10.1038/12419 Gendron FP Benrezzak O Krugh BW Kong Q Weisman GA Beaudoin AR Purine signaling and potential new therapeutic approach: possible outcomes of NTPDase inhibition Curr Drug Targets 2003 3 229 245 12041737 Westfall DP Todorov LD Mihaylova-Todorova ST ATP as a cotransmitter in sympathetic nerves and its inactivation by releasable enzymes J Pharmacol Exp Ther 2002 303 439 444 12388622 10.1124/jpet.102.035113 Roberto Meyer-Fernandes J Ecto-ATPases in protozoa parasites: looking for a function Parasitol Int 2002 51 299 303 12243784 10.1016/S1383-5769(02)00017-X Kannan S E-NTPase /E-NTPDase: a potential regulatory role in E-kinase/PKA-mediated CD36 activation Cell Biol Int 2003 27 153 163 12662972 10.1016/S1065-6995(02)00296-2 Marcus AJ Broekman MJ Drosopoulos JH Islam N Pinsky DJ Sesti C Levi R Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases J Pharmacol Exp Ther 2003 305 9 16 12649347 10.1124/jpet.102.043729 Linden J Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection Annu Rev Pharmacol Toxicol 2001 41 775 787 11264476 10.1146/annurev.pharmtox.41.1.775 Dhalla NS Yates JC Proveda V Calcium-linked changes in myocardial metabolism in the isolated perfused rat heart Can J Physiol Pharmacol 1977 55 925 933 902166 Dhalla NS Pierce GN Panagia V Singal PK Beamish RE Calcium movements in relation to heart function Basic Res Cardiol 1982 77 117 139 6284117 Zimmerman AN Hulsmann WC Paradoxical influence of calcium ions on the permeability of the cell membranes of the isolated rat heart Nature 1966 211 646 647 5968741 Alto L Elimban VE Lukas A Dhalla NS Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox Mol Cell Biochem 2000 207 87 94 10888231 10.1023/A:1007046316277 Gordon JL Extracellular ATP: effects, sources and fate Biochem J 1986 233 309 319 3006665 Mitchell CH Carre DA McGlinn AM Stone RA Civan MM A release mechanism for stored ATP in ocular ciliary epithelial cells Proc Natl Acad Sci U S A 1998 95 7174 7178 9618558 10.1073/pnas.95.12.7174 Aleu J Martin-Satue M Navarro P Lara IP Bahima L Marsal J Solsona C Release of ATP induced by hypertonic solutions in Xenopus oocytes J Physiol 2003 547 209 219 12562935 Bodas E Aleu J Pujol G Martin-Satue M Marsal J Solsona C ATP crossing the cell plasma membrane generates an ionic current in xenopus oocytes J Biol Chem 2000 275 20268 20273 10764752 10.1074/jbc.M000894200 Caldwell CC Hornyak SC Pendleton E Campbell D Knowles AF Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status Arch Biochem Biophys 2001 387 107 116 11368171 10.1006/abbi.2000.2216 Wang TF Ou Y Guidotti G The transmembrane domains of ectoapyrase (CD39) affect its enzymatic activity and quaternary structure J Biol Chem 1998 273 24814 24821 9733785 10.1074/jbc.273.38.24814 Abraham EH Sterling KM Kim RJ Salikhova AY Huffman HB Crockett MA Johnston N Parker HW Boyle WE JrHartov A Demidenko E Efird J Kahn SA Grubman DM Jefferson Robson SC Thakar JH Lorico A Rappa G Sartorelli AC Okunieff P Erythrocyte membrane ATP binding cassette (ABC) proteins: MRP1 and CFTR as well as CD39 (ecto-apyrase) involved in RBC ATP transport and elevated blood plasma ATP of cystic fibrosis Blood Cells Mol Dis 2001 27 165 180 11358378 10.1006/bcmd.2000.0357 Stout JG Kirley TL Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity Biochemistry 1996 35 8289 8298 8679585 10.1021/bi960563g Sperelakis N Schneider JA A metabolic control mechanism for calcium ion influx that may protect the ventricular myocardial cell Am J Cardiol 1976 37 1079 1085 1274870 10.1016/0002-9149(76)90428-8 Ziegelhoffer A Anand-Srivastava MB Khandelwal RL Dhalla NS Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase Biochem Biophys Res Commun 1979 89 1073 1081 227376 Schoffeniels E Dandrifosse G Protein phosphorylation and sodium conductance in nerve membrane Proc Natl Acad Sci U S A 1980 77 812 816 6928680 Kannan S Lalonde C Zahradka P Dhalla NS Molecular cloning of rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein) J Mol Cell Cardiol 1998 30 2261 2268 9925363 10.1006/jmcc.1998.0786 Ziegelhoffer A Dhalla NS Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation Mol Cell Biochem 1987 77 135 141 2963954 Buchweitz O Bianchi CP Myocardial magnesium transport: effect of gramicidin S and epinephrine Life Sci 1994 55 1853 1861 7526107 10.1016/0024-3205(94)90096-5 Romani A Dowell E Scarpa A Cyclic AMP-induced Mg2+ release from rat liver hepatocytes, permeabilized hepatocytes, and isolated mitochondria J Biol Chem 1991 266 24376 24384 1662210 Bianchi CP Liu D Calcium dependent magnesium uptake in myocardium Life Sci 1993 52 1225 1229 8450715 10.1016/0024-3205(93)90105-C Zhao D Dhalla NS Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase Arch Biochem Biophys 1988 263 281 292 2967673 Smith TM Kirley TL Glycosylation is essential for functional expression of a human brain ecto-apyrase Biochemistry 1999 38 1509 1516 9931016 10.1021/bi9821768 Stout JG Kirley TL Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity Biochemistry 1996 35 8289 8298 8679585 10.1021/bi960563g Picher M Boucher RC Human airway ecto-adenylate kinase. A mechanism to propagate ATP signaling on airway surfaces J Biol Chem 2003 278 11256 12264 12551890 10.1074/jbc.M208071200

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Theor Biol Med Model

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==== Front Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-1-41531223110.1186/1742-4682-1-4ResearchWe still fail to account for Mendel's observations Porteous John W [email protected] Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK2004 16 8 2004 1 4 4 10 6 2004 16 8 2004 Copyright © 2004 Porteous; licensee BioMed Central Ltd.2004Porteous; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The present article corrects common textbook accounts of Mendel's experiments by re-establishing what he wrote and how he accounted for his observations. It notes the long-established tests for the validity of any explanations that purport to explain observations obtained by experiment. Application of these tests to Mendel's paper shows that the arguments he used to explain his observations were internally consistent but were, on one crucial issue, implausible. The same tests are applied to the currently accepted explanation for Mendel's observations. Conclusions The currently favoured explanation for Mendel's observations is untenable. It misrepresents Mendel, fails to distinguish between the parameters and the variables of any system of interacting components, its arguments are inconsistent, it repeats the implausibility in Mendel's paper, fails to give a rational explanation for his observed 3:1 trait ratio and cannot explain why this ratio is not always observed in experimental practice. A rational explanation for Mendel's observations is initiated. Readers are challenged to complete the process before a further article appears. ==== Body 1. Background We all talk, more or less knowingly, about Mendelian genetics. But four questions need to be asked and answered. 1. Do we understand Mendel's work? To judge from nearly all modern accounts of genetics, we do not. Mendel's paper of 1866 has been persistently misrepresented ever since it was rescued from obscurity in 1900. 2. Do we teach our students a rational description of the inheritance of traits? The answer is again no. Why? Because our current depiction of the inheritance of traits or characteristics is based on false statements, inconsistent arguments and an implausible assertion. 3. Does the current description of Mendelian genetics account for his observations of dominant and recessive traits? No, for the reasons given in answering question 2. 4. Do we account rationally for Mendel's observation of a 3(dominant):1(recessive) trait ratio in some but not all of his experiments? The answer is again no. The reasons will become clear in this article and its successor. A survey of the relevant literature for the period from 1900 to 2003 shows that the various misrepresentations of Mendel's first paper [1] are of long standing. This is not the place to review all the accumulated historical evidence. The present article concentrates on demonstrating that the currently favoured depiction of elementary Mendelian genetics is untenable; it fails to achieve its intended purpose. A change in the concepts and notation for the interpretation (and teaching) of elementary genetics is suggested. There are two long-established tests of the validity of any hypothesis or proposed explanation for the results observed by experiment. The first test asks: Are all the arguments employed consistent, one with all the others? The second test asks: Are all the proposed mechanisms plausible? Could they be confirmed by experiment, i.e. by a "real"experiment or by a logical "thought experiment". Both tests must be passed if the proposed explanations for the observations are to be accepted. If judgement is being passed on work carried out in the distant past, allowance must be made for the availability or lack of availability of tests of plausibility at that time. On the other hand, we should not hesitate to criticise a current explanation that fails tests of plausibility that are now available but were not available in the past. These two tests of validity (consistency and plausibility) will be applied to Mendel's explanation for his observations and to the currently favoured explanation for his observations. We must first re-establish what experiments Mendel performed and what he wrote in his published accounts of these experiments in order to correct the various false textbook descriptions of Mendel's work. For this purpose it is necessary to study authentic reprints of his two papers [1,2]. The first paper is the one we are concerned with here; it was reprinted [3] and in a version [4] correcting several type-setting errors that occurred when Mendel's manuscript was set in typescript. The translation into English by Sherwood [5] avoided several errors in earlier attempts to translate Mendel's Versuche paper [1]. There may be other sound translations, but Sherwood's version is strongly recommended. It is accurate and also captures Mendel's literary style. 2. Mendel's experiments and his conclusions 2.1. Why did Mendel carry out his experiments? Many earlier biologists had noted the appearance of hybrid plants but their findings did not show how hybrids arose, whether there was any regularity in their occurrence, or how their properties were related to those of their parents. Mendel showed that there was a general rule for the appearance of hybrid plants and that an exact relationship existed between the traits displayed by hybrids and those displayed by their parents. Hence the title of his first paper: Versuche über Pflanzen-hybriden (Investigations on plant hybrids). 2.2. Mendel's preliminary work and his conditions for successful experimentation Mendel recognised five preconditions for success in his experiments on the origin of hybrids: (i) He needed suitable plants for his experiments. He chose Pisum sativum (the edible pea plant) for most of his work because many established varieties were readily available; and because the flowers enclose the reproductive organs, so minimising accidental cross-fertilisation by insect-or air-borne pollen. (ii) Pisum sativum, like all leguminosae, is androgynous. The flowers contain both male (pollen or sperm) and female (germinal or ova) cells and are therefore normally self-fertilising. This provided experimental advantages, as we shall see. (iii) It was necessary to have stocks of true breeding plants for his cross-fertilisation experiments. He therefore spent much time establishing that 22 varieties of edible pea plants were in fact true breeding. He discarded those plants that were not true breeding before starting his experiments on hybridisation. (iv) He had to ensure that any cross-fertilisations were strictly under his control. To achieve this control, he removed all the immature pollen-bearing stamens from a true-breeding pea plant that displayed a particular trait, e.g. green seeds, then transferred pollen to these emasculated flowers from another true breeding pea plant that displayed an alternative form of the same trait (e.g. yellow seeds). (v) Success depended on meticulous enumeration of the occurrence of hybrids, and of alternative traits, in the populations of plants that arose from his cross-and self-fertilisation experiments; and on repetition of each cross-fertilisation and self-fertilisation experiment in order to obtain reliable, average, results. Table 1 reveals the magnitude of Mendel's undertaking and records his observations on the occurrence of hybrids, and of plants displaying either dominant or recessive traits (see further descriptions in the following section). Reciprocal crosses gave the same results; Mendel thus established that male and female sex cells contributed equally to the final outcomes. Table 1 Mendel's novel observations summarised. Mendel demonstrated that crossing parental plants bearing alternative forms (A) and (a) of any one of seven traits generated a F1 population of plants (not shown) all of which were hybrids (Aa). Each of these F1 hybrid plants displayed only one of the two alternative parental traits, defined as the dominating trait (A). When these F1 hybrid plants were allowed to self-fertilise, the ratio of dominant to recessive traits in the F2 population was always close to 3:1. Pairs of parental plants Their F2 progeny Dominant traits (A) Recessive traits (a) Number of F2 plants examined Dominant:recessive trait ratio in the F2 population Green pods Yellow pods 580 2.82:1 Axial flowers Terminal flowers 858 3.14:1 Red flowers White flowers 929 3.15:1 Long stems Short stems 1064 2.84:1 Inflated pods Constricted pods 1181 2.95:1 Round seeds Wrinkled seeds 7324 2.96:1 Yellow seeds Green seeds 8023 3.01:1 2.3. Bateson's notation for successive stages in breeding experiments The following account uses the notation proposed by Bateson [6] for successive generations arising from sexual reproduction:- P = the original male and female parental generations; F1 = the first filial progeny population arising from crosses between plants of the P generation; F2 = the second filial generation that arises from sexual reproduction by members of the F1 generation – and so on. The advantage of Bateson's notation is that it does not depend on any preconceived ideas about the mechanisms of inheritance of traits during sexual reproduction. It can therefore be used to describe the stages in Mendel's experiments without misrepresenting any of his observations, arguments or conclusions. 2.4. Mendel's initial observations summarised Table 1 shows the results of seven different cross-fertilisations between parental (P) plants displaying alternative forms of the same trait, e.g. red rather than white forms of the trait "flower colour"; all individual plants in the F1 population displayed only one of the two parental trait forms. Also shown are the results observed by Mendel when he allowed these F1 plants to self-fertilise; the ratio of (A) form to (a) form plants was, in every case, close to 3:1. Mendel also carried out experiments in which he cross-fertilised plants displaying concurrently two or three trait differences, and then recorded the occurrence of each trait in the F1 and F2 generations. These results are not shown here but they were consistent with the findings exemplified in Table 1. These initial findings led Mendel to a remarkable generalisation and a definition. (i) All plants in the F1 population displayed only one of any two differing trait forms (A) and (a) displayed by the parental (P) plants. (ii) He defined the trait form that was displayed in the F1 plants as das dominirende Merkmal (A) – the dominating trait (A). He defined the alternative trait form, which did not appear in any of the F1 plants, as das recessivem Merkmal (a) – the recessive trait (a). 2.5. Further experiments Mendel now faced the problem of explaining how the 3(dominant):1(recessive) trait ratio arose in the F2 population of plants (Table 1). In further experiments on each of the seven crosses shown in Table 1, he was able to show that those F2 plants he had identified by the symbol (a) were 'constant form' (true-breeding) plants; i.e., when they were allowed to self-fertilise, all their F3 progeny displayed the same parental trait (a). On the other hand, when F2 plants initially identified by the symbol (A) were allowed to self-fertilise some proved to be 'constant form' plants because, when they were allowed to self-fertilise, they produced F3 progeny that again displayed this same parental trait (A). But other plants initially identified by the symbol (A) in the F2 population were not 'constant form' plants. Some of their F3 progeny did display the original parental trait (A). Other plants in the same F3 population displayed the alternative parental trait (a). Yet other plants in this F3 population were again not 'constant form' plants. They were like the F1 plants (their "grandparents") and like the F2 parents from which they were immediately derived. When they were allowed to self-fertilise, some of their progeny displayed the (A) form, some the (a) form of trait and some were again like the F1 plants. The experimental procedures Mendel used to make these distinctions are readily understood by reading a reprint of the original paper or a reliable translation. Given this ability to distinguish, by experiment, between those plants initially designated (A) and those now designated (Aa), Mendel was able to state the average distribution of trait forms among the plants of the F2 population as (one dominant: two hybrid: one recessive) or, in his notation, (A + 2Aa + a); i.e. the 3:1 trait ratio factored into the proportions 1:2:1. Mendel was now able to add a further generalisation: When F1 plants were allowed to self-fertilise, 1/4 of the F2 population displayed the 'constant form' parental trait (A) that was displayed by the F1 plants, 1/4 displayed the 'constant form' parental trait (a) that did not appear in any of the F1 plants (Table 1), and 1/2 were hybrids (Aa) that displayed only the dominant trait (A) but were not 'constant form' plants. 2.6. Mendel's notation Mendel used upper case and lower case italicised letters throughout his paper to denote, by definition, dominant and recessive traits. Examples have already been given of the use of letters (A) and (a) when only one trait difference between parental plants was tested (Table 1). Mendel made similar use of the letters (B) and (b), (C) and (c) when he described experiments in which two or three trait differences were displayed concurrently. For reasons given in Section 2.5 these single letters also designated what Mendel called 'constant forms' of traits. Plants displaying these traits were 'true-breeders'; they were the parental plants he used in cross-fertilisations (Table 1). There is one further crucial feature of Mendel's single letter notation for 'constant form' traits. These letters (A, a, B, b, C, c) did not represent the structure or composition of the traits. All the traits shown in Table 1 obviously had complex compositions. But, irrespective of such complexity, each dominant trait was denoted by (A) and each recessive trait by (a) in Table 1. The traits were what Mendel could see with his own eyes. He distinguished a dominant trait from a recessive trait by qualitative observations. He was not concerned with and did not analyse the structural composition of the traits. The letters (A, a, B, b, C, c) represented classes of traits – a dominant class represented by an upper case letter, and a recessive class of trait represented by the corresponding lower case letter (Table 1). It is necessary to recognise these facts if a rational explanation for Mendel's observation is to be obtained; and if gross misrepresentations of Mendel's paper are to be detected. Why then did Mendel use a combination of letters (e.g. Aa) to represent hybrid plants? This will become clear in section 2.7. 2.7. Postulates and arguments; Mendel's explanations of his observations Mendel accounted for the two generalisations (section 2.4) by the following postulates and arguments; they were based on his further experiments (section 2.5):- (1) All the F1 plants were hybrids (Aa) in welcher beide Merkmale vereinigt sind – in which both (parental) traits (A and a) were united; trait (a) was not displayed by these hybrids, so that these hybrids displayed what he had defined as the dominating trait (A) only. (2) The traits (A) and (a) in the F1 hybrids (Aa) segregated into traits (A) and (a) during formation of the male pollen (sperm) cells and also during formation of the female germinal cells (ova). Thus, each pollen cell and each germinal cell carried only one trait – either (A) or (a) but not both. (3) Fertilisation of one germinal cell by one pollen cell was a random event. (4) When a pollen cell bearing trait (A) fertilised a germinal cell bearing the same trait (A), all their progeny displayed the trait (A). Likewise, when a pollen cell bearing a trait (a) fertilised a germinal cell bearing the same trait (a), all their progeny displayed the trait (a). But when a pollen cell bearing trait (A) fertilised a germinal cell bearing the alternative trait (a), the resulting plant was the hybrid (Aa); if the pollen cell displaying a trait (a) fertilised a germinal cell displaying the alternative trait (A), the outcome was again a hybrid (Aa). In either event, the hybrid (Aa) displayed only the dominant trait (A). (5) Mendel illustrated these postulates and explanations in a diagram (Figure 1) showing the consequences of self-fertilisation of F1 hybrids (Aa), given that traits (A) and (a) in the hybrid (Aa) first segregated into individual pollen cells (sperm) and individual germinal cells (ova) before recombining, in random fashion, during formation of the F2 population. The arrows in Figure 1 represent the fertilising event. Figure 1 Mendel's diagrammatic explanation for the formation of the F2 population of plants produced by self-fertilisation of his F1 hybrids. Mendel proposed that F1 hybrids (Aa) contained a dominant trait (A) that was displayed and a recessive trait (a) that was not displayed. Self-fertilisation of F1 hybrids (Aa) then involved segregation of the component traits (A) and (a) into individual male pollen and female germinal cells, as shown in his diagram. Mendel proposed that if a male pollen cell carrying a trait (A) fertilised a female germinal cell carrying the same trait (A), the progeny would display trait (A). He used the analogous argument for the generation of progeny bearing trait (a). Only if male and female sex cells carried differing forms of a given trait (A or a but not both) would the progeny be hybrids (Aa). Thus random recombination of the segregated traits during self-fertilisation of hybrids would yield (on average) the F2 population of plants represented by the trait series (A + 2Aa + a) shown below Mendel's original diagram. Note two crucial points:- (i) Mendel observed and recorded the occurrence of traits (die Merkmale) or the characters (die Charaktere) in his plants and their seeds, not the mechanisms underpinning these occurrences. These mechanisms could not have been investigated in 1866. (ii) All Mendel's explanations were based solely on observations of the changes in the occurrence of alternative traits in successive populations that arose from cross-or self-fertilisations and back-crosses. 2.8. Comment Mendel was a well-trained scientist [7], an astute thinker, a careful and systematic experimentalist, an expert hybridiser and an exemplary writer but he was not the first geneticist. That title should go, possibly, to Bateson [6,8,9] for advocating Mendel's experimental methods, for showing that Mendel's findings could be repeated in animals, and for emphasising that combination, segregation and recombination of traits during gametogenesis was the most important feature of Mendel's work. Moreover, Bateson realised [[6]; in a footnote on page 133] that the occurrence of alkaptonuria, one of the "Inborn Errors of Metabolism" first reported by Garrod [10-12], was an example of Mendelian recessivity of a trait or character. Bateson, incidentally, coined the word "genetics". Another leading contender for the title "the first geneticist" was the Danish biologist, Johannsen [13,14], an equally enterprising experimentalist and astute thinker. Johannsen [14] was the first to define the term "das gen; (plural) die gene" as the determinant of a trait; he was also the first to make a clear distinction between the genotype (der Anlagetypus) and the phenotype (der Erscheinungstypus) on the basis of his experiments with self-fertilising bean plants. In Johannsen's experiments the weights of individual beans were the characteristics or traits. He had, in effect, repeated Mendel's experiments but by measuring a trait (individual bean weights in successive populations of plants) he was able to introduce three new concepts (gene, genotype and phenotype) that were the most significant, after Mendel's concepts of combination, segregation and recombination of traits during gametogenesis, in understanding the origin of genetic phenomena (the origin of changing traits). Failures to recognise the significance of Johannsen's work [13,14] prevented the development of rational concepts in genetics for at least the first two decades of the 20th century. This failure is, surprisingly, still evident in current depictions of elementary Mendelian genetics (Section 3). 2.9. The tests of validity applied to Mendel's explanation for his observations It is clear that Mendel's experimental procedures (sections 2.2, 2.5) were sound; his notation was simple, unambiguous and consistently applied (section 2.6). His arguments (section 2.7) for a combination of traits in forming the F1 hybrids (Aa) are consistent with his arguments for the segregation of the component traits of the hybrid into separate gametes, and their random recombination in generating the F2 population (A + 2Aa + a). Mendel's arguments pass the test of consistency. It is equally clear (but hitherto not noticed) that Mendel's explanations failed the test of plausibility. Mendel postulated that a F1 hybrid (Aa) was formed by combining the two differing traits (A) and (a) of their parents. He did not explain how a F1 hybrid (Aa) displayed only trait (A) and how it did not display trait (a), even when some F2 plants, like one of the two original parental (P) plants, did display trait (a). What explanation could we now give for this selective display of only one of two traits that are said to be combined in a hybrid? It may be (and has been) argued by some that trait (A) was displayed by the hybrids (Aa) because (A) was a dominant trait and (a) was a recessive trait. Such statements do not even qualify as a circular argument. They are illogical. Such statements fail to distinguish between an arbitrary definition and a plausible explanation. Mendel's definition of a dominant trait should be seen as an arbitrary device that accounts for his observation (by experiment) that his hybrids (Aa) in the F2 populations (A + 2Aa + a) displayed trait (A) but not trait (a). A word of caution is necessary. Mendel's formulation (Aa) for a hybrid was crucial in establishing his consistent arguments; it was also the basis for Bateson's recognition that the essential features of Mendel's work were the concepts of combination, segregation and recombination of alternative traits (i.e., components of the phenotype). If we now wish to replace Mendel's implausible formulation (Aa) for a hybrid by a plausible formulation, we face the prospect of abandoning the rest of Mendel's arguments. That is not to say that we abandon admiration for Mendel's work. For its time, it was unsurpassed and should be recognised as one of the important steps in the development of experimental procedures in what became known as genetics. We should take care not to misrepresent Mendel's experiments and his arguments. It will become clear that misrepresentations of Mendel's paper have served only to sustain untenable concepts in current biology. In the post-Mendel era we assert that it is not components of the phenotype that segregate and recombine. It is the alleles (i.e., components of the genotype) that combine, segregate and recombine. May we then anticipate that modern explanations of Mendel's observations will pass the tests of consistency and plausibility? 3. Current accounts of elementary Mendelian genetics 3.1. Explanations of Mendel's observations The currently favoured explanation for Mendelian heredity in general, and in particular for the occurrence of Mendel's 3(dominant):1(recessive) trait ratio, is shown in Figure 2. Figure 2 The currently favoured depiction of Mendelian inheritance following self-fertilisation of F1 hybrids represented by the allele pair (Aa). Section 3.1 of the text records the arguments commonly used in attempts to account for the alleged F2 trait series (AA + 2Aa + aa) and for Mendel's 3(dominant):1(recessive) trait ratio. Sections 3.2 and 3.3 discuss the faults in these arguments. The assertions and descriptions generally attached to Figure 2 are as follows. (i) Mendel explained his experimental results by assuming that particles or factors (now called alleles) determined or specified the observed traits. (ii) (A) is a dominant allele;(a) is a recessive allele. (iii) The alleles in the male and female heterozygous somatic cells (Aa) segregate into separate gametes. Each gamete then contains only one dominant allele (A) or only one recessive allele (a). (iv) Fertilisation is a completely random event. Given a large number of fertilisation events, the possible recombinations of alleles are those displayed in the four squares. (v) Therefore the average distribution of the alleles at one diploid locus in the resulting progeny population of individual plants will be (AA + 2Aa + aa). It is then argued that: (vi) The dominant allele pair (AA) will give rise to a dominant trait (AA). (vii) The recessive allele pair (aa) will give rise to a recessive trait (aa). (viii) In the heterozygote (Aa), the recessive allele is ineffective, or is suppressed by the dominant allele (A), so that only the dominant allele (A) is expressed in the heterozygote. Expression from one (A) is as effective as that from two dominant alleles (AA). Thus the heterozygote (Aa) expresses a dominant trait. (ix) Therefore the allele series (AA + 2Aa + aa) is expressed (in a population of the progeny plants, animals or cells) as the trait series (AA + 2Aa + aa). (x) This trait series gives rise to Mendel's 3(dominant):1(recessive) trait ratio (by the arguments in vi, vii, viii). 3.2. Faults in these currently favoured descriptions of Mendelian genetics There are seven faults in the descriptions and arguments attached to Figure 2. (i) Mendel is misrepresented; he did not assume that particles or factors specified the observed traits. It is historically inaccurate and scientifically misleading to suppose that he made any such assumption. (ii) The letters (A) and (a) are Mendel's notation for dominant and recessive traits (Figure 1, Table 1). If we are to continue to discuss Mendelian genetics, these notations (and the nomenclature dominant and recessive) should refer to traits alone. (iii) Figure 2 fails to distinguish between the components of the genotype and the components of the phenotype (Johannsen, Section 2.8) because it asserts that alleles are dominant or recessive; and uses the same notation (A and a) and the same nomenclature (dominant and recessive) for both. (iv) Because we must not confuse alleles with traits, we could reasonably write an allele series as (UU + 2Uu + uu); this states that a given locus, in three genetically related diploid cells, comprises a pair of two normal alleles (UU), or one normal and one mutant allele (Uu), or a pair of two mutant alleles (uu). Mutations change the allele constitution or composition at a locus. The modern (non-Mendelian) notation (AA + 2Aa + aa) in Section 3.1 (items vi, vii) then states explicitly that a dominant trait (AA) comprises two aliquots (A + A) of some material substance or of two doses of dominance (A + A); likewise that a recessive trait (aa) is composed of two entitities (a + a) or two doses of recessivity. This is simply not true. It was not true in Mendel's time and it is not true today. Furthermore, it is not what Mendel's notation meant. It was pointed out (Sections 2.5, 2.6) that Mendel's notation (A) and (a) distinguished classes of traits, specifically 'constant form' classes of traits (Table 1). To substitute (AA) for (A) and (aa) for (a) in a trait series is illogical and indicates a regrettable failure to read Mendel's paper with the care that should be given to one of the classic papers in biology. (v) If the arguments attached to the homozygotes in Figure 2 are sound, they should also apply to the heterozygote. It is argued in Figure 2 that two dominant alleles (AA) generate a dominant trait (AA); and that two recessive alleles (aa) generate a recessive trait (aa). In other words, it is asserted that there is a direct, positive, linearly proportional (or additive) relationship between the allele constitution at a gene locus and the constitution of the trait expressed from that locus. If we are to be consistent, the same arguments should apply to the heterozygote (Aa). On the contrary, the arguments in section 3.1 (item viii) state that one dominant allele (A) in a heterozygote (Aa) is as effective as two dominant alleles (AA) in the homozygote. The arguments in item (viii) are therefore inconsistent with arguments in items (vi) and (vii). Item (viii) also transfers Mendel's implausible assertion that a hybrid (Aa) displays only trait (A) to the equally implausible assertion that one allele (A) in a heterozygote (Aa) is as good as two such alleles in a homozygote (AA). The argument in item (viii) that allele (a) is ineffective is an extreme case; it is therefore not generally applicable. The alternative argument, that allele (a) in a heterozygote is suppressed by the dominant allele (A), lacks any experimental support or rational theoretical justification. Items vi, vii and viii attached to Figure 2 are arbitrary, irrational and implausible devices applied to the heterozygote alone; they seem to have been introduced solely in order to arrive at the desired result. (vi) Figure 2 and the attached arguments thus fail to give rational explanations for the occurrence of dominant and recessive traits and for Mendel's 3(dominant):1(recessive) trait ratio. (vii) Figure 2 does not and cannot account for the observation that dominance and recessivity are not observed for all traits. The assertion in Figure 2 that the alleles are themselves "dominant" or "recessive" (and thus determine that traits are dominant or recessive) conflicts with inability of Figure 2 to explain why dominant and recessive traits are not always observed; nor does Figure 2 account for the observation that, when dominance and recessivity do occur, they do not always exhibit a 3:1 trait ratio. 3.3. Comments on these faults It is necessary to restate fault (iii) in section 3.2 in more widely applicable terms. It is illegitimate to use the same notation and nomenclature for a parameter and a variable in the same system. Parameters are those components of any system that are directly accessible to the experimentalist; they can be changed and maintained by the experimentalist at the new value, at least for the duration of an experiment. Variables are those components of the same system that are not directly accessible to the experimentalist; they can be changed and maintained at a new value only by making a finite change in at least one parameter of the system or of its immediate environment. The magnitudes of individual variables, in any system, respond to changes in the magnitude of one or more parameters of the system or of the immediate environment. In the case under discussion, the alleles are parameters (and part of the genotype); the traits are variables (and part of the phenotype). If the parameters and variables of any system of interacting components are represented by the same notation and the same nomenclature, confusion will inevitably result – as illustrated by Figure 2, by the assertions (i) and (ii) and by the false arguments (vi) to (x). Traits may be dominant or recessive [1]; alleles cannot also be dominant or recessive. Figure 2, and the arguments attached to it, fail all tests of consistency and plausibility (Section 2.9); they also fail the test of historical accuracy. 3.4. Another example of the improper transfer of dominance/recessivity from traits to alleles The primary error in Figure 2 is the illegitimate transfer of Mendel's terms "dominant" and "recessive" from traits (variables) to alleles (parameters), followed immediately by the reverse (and perverse) argument that the traits specified by the alleles must be dominant or recessive because the alleles are dominant or recessive. This habit is unscientific. It also occurs in discussion of mutations of non-catalytic proteins. When haemoglobin A (HbA) is mutated to the sickle cell haemoglobin (HbS), the three possible trait forms are correctly depicted as follows: (A/A) – the homologous, normal/normal protein, condition; (A/S) – the heterologous, normal/mutant protein (sickle cell), condition; (S/S) – the homologous, mutant/mutant protein, condition. Contrast these depictions with those sometimes found: (A/A) – the dominant condition; (A/S) – the sickle cell condition; (S/S) – the recessive condition. These latter statements depend solely on the illegitimate transfer of Mendel's terms dominant and recessive from traits (variables) to alleles (parameters) and the contention that, if alleles are themselves dominant or recessive, their expressed traits must always be dominant or recessive. If changes in the composition of non-catalytic proteins do explain the occurrence of Mendel's dominant and recessive traits, we require a demonstration that does not depend on these illogical notions. The sickle cell trait (A/S) in humans is significantly different from the normal trait (A/A). Those carrying the sickle cell (A/S) condition enjoy an advantage in areas where malaria is endemic. They do not die from malaria as frequently as those in the population with the (A/A) condition. The sickle cell condition (A/S) is debilitating but, provided it is not too debilitating, the frequency in the local population of those carrying the (A/S) protein pair is greater than it would be in malaria-free areas. This higher frequency of the sickle-cell (A/S) condition in areas where malaria is endemic is often said to be an example of "over-dominance". The term "over-dominance" is inappropriate. It presumably arose from the illegitimate transfer of the terms dominant and recessive noted above. The appropriate term is "heterozygous superiority". The "superiority" indicates the better chance of surviving in regions where malaria is endemic. 4. Conclusions: beginning a rational explanation for Mendel's observations The illegitimate use in Figure 2 of the same notation (A and a), and the same nomenclature (dominant and recessive), to describe an allele series and a trait series can be traced to Sutton [15]. Sutton asserted that the proportions of the chromosome pairs in the F2 population "would be expressed by the formula AA:2Aa:aa which is the same as that given for any character in the Mendelian case." Mendel's expression (A + 2Aa + a) gave the proportions of characters in his F2 population as A:2Aa:a. Sutton gave no justification for rewriting these proportions in the form AA:2Aa:aa. By writing the expression for chromosome pairs as AA:2Aa:aa and the expression for the proportions of F2 characters as AA:2Aa:aa, Sutton established a direct, one-for-one, relationship between pairs of chromosomes and the traits arising from them. This false relationship also persists in the currently favoured depiction of Mendelian genetics (Figure 2). Sutton's notation for pairs of chromosomes (AA:2Aa:aa) was later transferred to pairs of alleles (what Sutton described as subunits of the chromosomes). It would be easy to blame Sutton for our present confusions. We should remember that Sutton, and those in the early years of the 20th Century who copied his error, were struggling to understand the hereditary origin of traits. We may more reasonably ask: Why, one hundred years later, are these obvious errors still one of the features of Figure 2? Have these errors not been noticed before or, if they have been noticed, why they have not been corrected? Why also has the inconsistency and the implausibility of the arguments attached to Figure 2 not been noticed or corrected? Why (in both of the examples given in sections 3.3 and 3.4) are alleles (components of the genotype) not distinguished, as they surely should be in genetics, from traits (components of the phenotype) by using different notations and nomenclatures for each? Traits (variables) may be dominant or recessive, as defined by Mendel. Alleles (parameters) are, always have been, and can only be normal or abnormal (mutant). Harris (pages 143–157 in reference [16]), for example, referred consistently to normal and abnormal alleles (not to dominant and recessive alleles), whereas, as noted above, alkaptonuria was a Mendelian recessive trait or character (page 133 in reference [6]; page 19 in reference [16]). A review of 13 textbooks of genetics showed that in 12 instances, dominance and recessivity were defined specifically as properties of genes or alleles. These texts, published between 1982 and 2002, were intended for student use; their definitions of dominance and recessivity ignore Mendel's definition of dominance and recessivity as properties of the traits (sections 2.4, 2.5, 2.6, 2.7); they take no account of the need to distinguish between the parameters and variables of a system of interacting components (section 3.2). In one of these 12 texts, it was further claimed that: "Mendel proposed the existence of what he called particulate unit factors for each trait". In another, that: "Mendel realised that some genes (dominant genes) expressed themselves when present in only one copy". In a third that: "Mendel imagined that during the formation of pollen and egg cells, the two copies of each gene in parents segregate". Of these three quoted texts: The first misrepresented Mendel; he did not "propose the existence of particulate unit factors for each trait". The second misrepresented Mendel by transferring his term dominirende ("dominating") from traits to genes; the second and the third quoted texts ignored the fact that the term "das gen" (plural "die gene") was first used and its role as the determinant of traits postulated by Johannsen, 43 years after Mendel's paper was published (Section 2.8); Mendel did not mention the word gene (Section 3.2). Of the 13 texts examined, only one gave a definition of dominance and recessivity that would have been recognised by Mendel. Even so, this author contradicted his correct definition of dominance and recessivity as properties of components of the phenotype by giving an explanation of elementary Mendelian genetics that employed Figure 2 and its associated arguments. All 13 of the texts examined ignored or contradicted the verifiable historical evidence (sections 2.2–2.7) and failed to make the obligatory distinction between the functions of alleles and the properties of traits. The correct nomenclature for alleles used by Harris (pages 143–157 in reference [16]) is, unfortunately, rarely if ever employed by other authors. Pasternak [17], for example, accepted that "in strict genetic terms, dominance and recessivity are descriptions of the phenotype and not of the genes." but then continued: "However, few textbooks bother to make the distinction, because it was both convenient and highly ingrained for geneticists and others to refer to dominant and recessive alleles." Ingrained it may be, convenient (and scientifically legitimate) it is not. If we continue to propose Figure 2 and the attached arguments as an explanation of Mendel's work, we deceive ourselves and encourage irrational thinking in our students at a time in their education when they are most vulnerable. It is extraordinary that an "explanation", like Figure 2, should still be found in textbooks intended for student instruction; it exposes our own confusion but explains nothing of scientific value in genetics. Any student who criticised Figure 2 and the attached arguments in an answer to an examination question would have shown commendable scientific insight but, according to current teaching, would be deemed to have failed that question. Barker [18], writing on another topic, suggested that it might take 50 rather than 25 years for textbooks "to get it right". On the evidence presented here, Barker was too optimistic. The four errors introduced by Sutton [15] remain uncorrected (Figure 2) 100 years later. To be fair to authors of textbooks of genetics, every author inevitably relies on what has been written by preceding authors. However that may be, we are faced with an uncomfortable question. Are we content to continue to deceive ourselves, to give our students a false picture of what Mendel achieved, and to provide them with untenable 'explanation' of his remarkable observations (Figure 2)? Presumably not, especially when we can very easily begin, in this article, to construct a rational explanation for Mendel's observations and for other observations of current interest in genetics. A fresh approach to the origins of dominant and recessive traits is needed. As a first step, we need to represent normal and mutant alleles by symbols that differ from those (A, a, B, b, C, c) used by Mendel to represent traits. We must replace symbols (A and a) for alleles in Figure 2 by quite different symbols; e.g. (U) to represent a normal allele, not a "dominant allele"; and (u) to represent a mutant or abnormal allele, not a "recessive allele". The F2 allele series in Figure 2 would then be, on average, UU + 2Uu + uu. Similarly, the trait series in Figure 2 must be replaced by Mendel's notation (A + 2Aa + a) because, as explained earlier, Mendel was concerned (as we are, first and foremost) only with understanding the origin of two classes of trait – the dominant class (A) and recessive class (a). We will later be concerned with the quantitative composition of traits. We have, however, already identified an implausibility in Mendel's notation (Aa) for a hybrid that, allegedly, displayed the trait (A). An implausibility, like an inconsistency, must be eliminated if we are to arrive at an internally consistent and plausible account of Mendel's observations. The implausible notation (Aa) can be eliminated by replacing it by the single symbol (H) for a hybrid. We have now adopted a stance that, in sharp contrast to Figure 2, distinguishes clearly between determinants and that which is determined. We have allocated a nomenclature and notation for alleles that is distinct from that allocated to traits. We have differentiated clearly between the parameters of the system (in this particular case, the components of the genotype) and the variables of the system (in this particular case, the components of the phenotype). Mendel found, by experiment, that the proportions of different plant forms in his F2 populations were 1(dominant trait):2(hybrids):1(recessive trait) or, in his notation, (A + 2Aa + a). Replacing Mendel's notation (Aa) for a hybrid by the single symbol (H) does not alter Mendel's experimental observation of the proportions of trait forms in the F2 populations (section 2.5). It does mean that we can avoid Mendel's implausible postulate that, although recessive trait plants did display trait (a), his hybrids (Aa) did not. We have, of course, to discover an experimentally verifiable mechanism that would explain why hybrids (H) display a trait that is sometimes indistinguishable and sometimes distinguishable from trait (A). Our remaining task is to explain rationally how this series of normal and mutant alleles (UU + 2Uu + uu) in the F2 population is expressed as the trait classes (A + 2H + a) in that population, where all that we have done is to replace Mendel's implausible (Aa) by a plausible (H). Note also that we have now also eliminated the illegitimate use of paired symbols for Mendel's dominant (A) and recessive (a) traits. Most of the clues that facilitate this task are present in this article. One clue is missing, but it can be inferred by asking how one allegedly dominant allele (U) in a heterozygote (Uu) could be as effective as two such alleles (UU) in a homozygote. A further article will provide the answers, but in the interval readers may like to rise to the challenge of explaining: (1) how dominant and recessive traits arise from normal and mutant alleles, and (2) why Mendel's 3:1 trait ratio, though not uncommon, does not always occur. ==== Refs Mendel G Versuche über Pflanzen-Hybriden Verhandlungen des Naturforschenden Vereines in Brunn 1866 4 3 47 Abhandlungen Mendel G Über einige aus kunstlicher befructung gewonnen Hieracium Bastarde Verhandlungen des Naturforschenden Vereines in Brunn 1869 8 26 32 Abhandlungen Flora A reprint of Mendel's "Versuche über Pflanzen-Hybriden" of 1866 1901 89 364 403 Křýženecký J Sosna M Fundamenta Genetica The revised edition of Mendels' classic paper with a collection of 27 original papers published during the rediscovery era 1965 Prague: Czechoslovak Academy of Science Sherwood ER Stern C, Sherwood ER Experiments on Plant Hybrids The origin of genetics. A Mendel source book 1966 San Francisco and London: W. H. Freeman and Company 1 45 Bateson W Reports to the Evolution Committee of the Royal Society London; Report No 1, Part III 1902 125 160 Reprinted in Reference [4], pp. 242–275 Mayr E The Growth of Biological Thought 1982 Cambridge, Mass, USA: Harvard University Press Bateson W Mendel's Principles of Heredity A Defence 1902 Cambridge: Cambridge University Press Bateson W Mendel's Principles of Heredity 1909 Cambridge: Cambridge University Press Garrod AE A contribution on the study of Alkaptonuria Proc Roy Med Chir Soc 1899 11 130 135 Garrod AE About Alkaptonuria Lancet 1901 ii 1484 1486 10.1016/S0140-6736(01)74537-0 Garrod AE The incidence of alkaptonuria: a study of chemical individuality Lancet 1902 ii 1616 1620 10.1016/S0140-6736(01)41972-6 Johannsen W Uber Erblichkeit in Populationen und reinen Linien Ein Beitrag zur Beleuchtung Schwebender Selektionsfragen 1903 Jena: Gustav Fischer Johannsen W Elemente der exakten Erblichkeitslehre mit Grundsatzen der biologische Variationsstatistisk 1909 Jena: Gustav Fischer Verlag Sutton WS The chromosomes in heredity Biol Bull Woods Hole 1903 4 231 251 Harris H The 'Inborn Errors' Today Garrod's Inborn Errors of Metabolism 1963 London: Oxford University Press 120 197 Pasternak JJ An introduction to Human Molecular Genetics Mechanisms of Inherited Diseases 1999 Bethesda, Maryland, USA: Fitzgerald Science Press Barker J When will they ever learn? Biologist 2002 48 244

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==== Front Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-1-51531223010.1186/1742-4682-1-5ResearchInvestigation of hydrophobic moment and hydrophobicity properties for transmembrane α-helices Wallace James [email protected] Onkabetse A [email protected] Frederick [email protected] David A [email protected] Department of Physics, Astronomy and Mathematics, University of Central Lancashire, Preston, PR1 2HE, UK2 Department of Forensic and Investigative Science, University of Central Lancashire, Preston, PR1 2HE, UK3 The Dean's Office, Faculty of Science; University of Central Lancashire, Preston, PR1 2HE, UK2004 16 8 2004 1 5 5 11 8 2004 16 8 2004 Copyright © 2004 Wallace et al; licensee BioMed Central Ltd.2004Wallace et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Integral membrane proteins are the primary targets of novel drugs but are largely without solved structures. As a consequence, hydrophobic moment plot methodology is often used to identify putative transmembrane α-helices of integral membrane proteins, based on their local maximum mean hydrophobic moment (<μH>) and the corresponding mean hydrophobicity (<H>). To calculate these properties, the methodology identifies an optimal eleven residue window (L = 11), assuming an amino acid angular frequency, θ, fixed at 100°. Using a data set of 403 transmembrane α-helix forming sequences, the relationship between <μH> and <H>, and the effect of varying of L and / or θ on this relationship, was investigated. Confidence intervals for correlations between <μH> and <H> are established. It is shown, using bootstrapping procedures that the strongest statistically significant correlations exist for small windows where 7 ≤ L ≤ 16. Monte Carlo analysis suggests that this correlation is dependent upon amino acid residue primary structure, implying biological function and indicating that smaller values of L give better characterisation of transmembrane sequences using <μH>. However, varying window size can also lead to different regions within a given sequence being identified as the optimal window for structure / function predictions. Furthermore, it is shown that optimal periodicity varies with window size; the optimum, based on <μH> over the range of window sizes, (7 ≤ L ≤ 16), was at θ = 102° for the transmembrane α-helix data set. Hydrophobic momentwindow sizeangular frequencytransmembrane proteinα-helix ==== Body Background Integral membrane proteins are the primary choice as targets when developing new drugs and although clearly of medical relevance, forming 20% – 30% of the gene products of most genomes, these proteins have been structurally determined in only about thirty cases [1,2]. Where high levels of sequence homology exist, an unknown protein's structure and hence, the location of its membrane interactive segments, can sometimes be deduced by direct comparison to known protein structures. However, where sequence information alone is available, the identification of transmembrane α-helical structure requires a bioinformatics approach to understanding the structure / function relationships of these α-helices. A number of α-helical properties have been used as models to study transmembrane α-helices and their structure / function relationships but the most commonly used are those based on the amphiphilicity of protein α-helices with the hydrophobic moment used as a measure of amphiphilicity [3]. To quantify the amphiphilicity of protein secondary structures, Eisenberg and co-workers [4] introduced the hydrophobic moment, μ(θ), which provides a measure of the structured partitioning of hydrophilic and hydrophobic residues in a regular repeat structure of period θ. For a structure comprising L consecutive residues, the general form of μ(θ) is given by: where Hj is the hydrophobicity of the jth residue within the sequence, and θ is the angular frequency of the amino acid residues forming the structure. Eisenberg et al., [4] assumed that for an α-helix, θ is fixed at 100°, and that a segment of eleven consecutive residues, equivalent to three turns of an α-helix, could be used to represent amphiphilic α-helices. These assumptions led to the more generally used measure of α-helix amphiphilicity, the mean hydrophobic moment <μH>, where <μH> = μ(100°)/11 As a major extension to the use of the hydrophobic moment, Eisenberg et al., [5] introduced hydrophobic moment plot methodology, which provides a graphical technique for the general classification of protein α-helices. Using this methodology, a putative protein α-helix is characterised according to its maximum <μH> and corresponding mean hydrophobicity, <H>, where this is defined by: The parameters <μH> and <H> are then plotted on the hydrophobic moment plot diagram (figure 1) and the location of the resulting data point used to classify the putative α-helix. Figure 1 Conventional hydrophobic moment plot analysis of the transmembrane protein data set. Figure 1a shows the hydrophobic moment plot diagram [5] with protein classification boundaries. Figure 1b shows the results of hydrophobic moment plot analysis of the 403 transmembrane sequences of our data set using the conventional values of L = 11 and θ = 100° [4]. The mean hydrophobic moment is widely used and generally regarded as a good predictor of α-helix amphiphilicity but the results of statistical analyses have shown the efficacy of hydrophobic moment plot methodology as a predictor of α-helical class to be less certain [6]. A number of authors have observed that the methodology can erroneously classify α-helices in cases where the hydrophobic moment for a particular amino acid sequence is greatly affected by the spatial arrangement of a few extreme amino acids, thus masking the overall nature of an α-helix [3]. However, a more fundamental source of erroneous classification could come from the questionable assumption made by hydrophobic moment methodology with respect to angular periodicity. It is known that in naturally occurring α-helices, θ can vary over the range (95° ≤ θ ≤ 105°) and between consecutive residues [7]. Clearly, assuming a fixed value of θ = 100° for all α-helices is an approximation and could lead to classification difficulties for the methodology. Furthermore, classification difficulties could arise from the arbitrary choice of window length made by the methodology as window length is known to have a profound effect on the relationship between <μH> and <H>[7]. It would seem that the optimisation of θ and window length are crucial to the classification of amphiphilic α-helices yet the values chosen for these parameters by hydrophobic moment plot analysis are not optimal for the classification of any single subclass. A number of studies have considered the significance of <μH> in relation to structure / function relationships of the α-helical classes described by hydrophobic moment plot methodology with common examples including: surface active α-helices, transmembrane α-helices and oblique orientated α-helices [8-10]. However, if different α-helical classes have differing optima for θ and window length, not only does this question the validity of results obtained in these studies but also questions the validity of α-helix classification according to hydrophobic moment plot methodology. In this paper we examine the criteria upon which the methodology is based and, in view of their medical relevance, we use transmembrane α-helices as a test data set. These α-helices possess central regions, which are predominantly formed by hydrophobic residues and interact with the membrane lipid core, and end regions, which are primarily formed by hydrophilic residues and reside in the membrane surface regions [8]. For the α-helices of our data set, we analyse the relationships for the mean hydrophobic moment and window size, angular frequency and the robustness to varying angular frequency. Correlations between the mean hydrophobic moment and mean hydrophobicity of transmembrane α-helices are established, verified and analysed to appraise biological function using resampling Bootstrap and Monte Carlo techniques [11,12]. Results A data set of 84 transmembrane proteins were identified within Swiss-Prot and used to generate a set of 403 transmembrane sequences (see Additional file 1). All sequences within the data were of 21 residues in length and showed less than 5% homology (data not shown). For the sequences of this data set, the maximum mean hydrophobic moment, <μH>, and its corresponding mean hydrophobicity, <H>, were determined and used to generate the hydrophobic moment plot shown in figure 1, based on the generally used 11 residue window (L = 11) introduced by Eisenberg et al., [4]. It can be seen that data points representing the sequences of our data set cluster around the transmembrane region identified by Eisenberg et al., [5] but as previously noted [6] there are a significant number that fall outside the boundaries of this region. In particular, many of this number possess <H> values less than 0.5 and would not be classified as transmembrane α-helices according to the hydrophobic moment plot taxonomy of Eisenberg et al., [5]. Even allowing for the diffuse nature of these boundaries on the hydrophobic moment plot diagram [5], these results clearly question the efficacy of hydrophobic moment methodology for the prediction of transmembrane α-helices. The above analysis was repeated except that window sizes varying in the range (7 ≤ L ≤ 20) were employed. The values for <μH> and corresponding <H> were plotted as above and the results for window sizes of 7, 9, 16 and 20 are shown in figure 2. It can be seen that a weak negative correlation exists between <μH> and <H> for smaller window sizes but that the level of correlation appears to reduce as window size increases. The sample correlation coefficients for the various window sizes are given in table 1. To conduct standard statistical tests to determine whether the population correlation coefficients do differ from zero, it is necessary to establish if these data are bivariate Normal. The P-values obtained from Anderson-Darling and Kolmogorov-Smirnov tests for Normality for the various window sizes with θ = 100° are shown in table 2. These results present clear evidence that the populations for the variates for each window size are not bivariate Normal. These findings prompted the use of the bootstrap procedures to estimate the confidence intervals for the population correlation coefficient values for the window sizes in the range (7 ≤ L ≤ 20). Figure 2 Hydrophobic moment plot analysis of the transmembrane protein data set with varying window size. Figure 2 shows the 403 transmembrane sequences of our data set, which were analysed according to hydrophobic moment plot methodology but with varying window size (L). In comparison to L = 1 (figure 1b), here in figure 2a, L = 7; in figure 2b, L = 9; in figure 2c, L = 16; and in figure 2d, L = 20. In each case, θ = 100°. Table 1 Sample correlation coefficients between <μH> and <H> for window sizes (7 ≤ L ≤ 20). Window size (L) Sample correlation coefficient (r) Window size (L) Sample correlation coefficient (r) 7 -0.57648 14 -0.34654 8 -0.45020 15 -0.31280 9 -0.30316 16 -0.17998 10 -0.40110 17 -0.15074 11 -0.47663 18 -0.21843 12 -0.33693 19 -0.20038 13 -0.30354 20 -0.15653 Table 2 Confidence Intervals for regression coefficient from bivariate Normality goodness-of-fit for window size L. * 93% Confidence Interval Window size (L) 95% Confidence Interval 99% Confidence Interval 7 (1.077, 1.112) (1.072, 1.176) 8 (1.051, 1.084) (1.046, 1.089) 9 (1.067, 1.091) (1.061, 1.095) 10 (1.091, 1.171) (1.078, 1.184) 11 (1.078, 1.134) (1.068, 1.149) 12 (1.046, 1.075) (1.042, 1.080) 13 (1.054, 1.110) (1.047, 1.167) 14 (1.055, 1.124) (1.044, 1.135) 15 (1.050, 1.087) (1.044, 1.093) 16 (1.036, 1.045) (1.030, 1.051) 17 (0.976, 1.001) (0.977, 0.999)* 18 (0.959, 0.980) (0.956, 0.983) 19 (0.957, 0.967) (0.955, 0.968) 20 (0.950, 0.960) (0.948, 0.962) The results of this investigation for θ = 100° are presented in figure 3. It would appear that the smaller window sizes do show correlations between <μH> and <H> and if this reflects a biological property of transmembrane sequences, it could be of use in the analysis and prediction of these motifs. It is known that angular frequency for a transmembrane α-helix varies between 95° and 107° [16], rather than being fixed at 100° as proposed by the methodology of Eisenberg et al., [4]. For each window size in the range (7 ≤ L ≤ 21) residues, to accommodate the findings of Cornette et al., [16], the fixed value of θ was therefore varied from 95° to 108° in increments of 1°. Once the optimal window had been obtained, to observe the discriminating effect of θ on <μH>, the <μH> values, denoted by Σ<μH>, were summed for the 403 sequences for each θ. Figure 4 shows the optimal θ, based on the maximum values of Σ<μH> for each window length. It can be seen that as the window size increases the total <μH> reduces approximately linearly until the intermediate size of eleven residues in length. For subsequent larger window sizes, we observe a further near linear reduction trend but at a reduced rate. The optimal angular frequency corresponding to each window size (7 ≤ L ≤ 21) is also given in figure 5. The overall relationship between Σ<μH>, the window size, L, and the angular frequency, θ, is finally depicted in figure 6 as a response surface diagram. Figure 3 Confidence intervals for the Correlation Coefficient. Figure 3a shows the 99% BCa confidence intervals for the correlation coefficients estimated from 4000 bootstrap replicates. Figure 3b shows the 99% ABC confidence intervals for the correlation coefficients. Figure 3c shows the 99% Delta Method confidence intervals for the correlation coefficients. Figure 4 Σ<μH> for the transmembrane protein data set for variable window sizes with optimised angular frequency. Figure 4 shows the variation of Σ<μH> for the 403 transmembrane sequences of our data set with window size (7 ≤ L ≤ 20) for optimised θ (95° ≤ θ ≤ 108°). Figure 5 The variation of optimal angular frequency with window size for the transmembrane protein data set. Figure 5 shows the variation of optimal angular frequency, θ, (95° ≤ θ ≤ 108°) with window size (7 ≤ L ≤ 20) for the 403 transmembrane sequences ofour data set Figure 6 Response surface diagram for the transmembrane protein data set. Figure 6. Response surface diagram for the Σ<μH> for window sizes (7 ≤ L ≤ 20) and angular frequency (95° ≤ θ ≤ 108°). To assess the robustness of <μH> to this fixed angular frequency assumption, and thus, the accuracy of the hydrophobic moment plot analysis for candidate transmembrane sequences, Monte Carlo simulation studies were conducted. Initially, the angular frequency, θ, was assumed to have a mean value, E(θ), fixed at 100° and the angle for each successive residue varied about E(θ). The random variation, X, followed a Normal distribution and six separate simulations were undertaken with X~N(100, σ2), where the standard deviation, σ, was set at 0.1°, 0.3°, 0.5°, 0.7°, 0.9° and 1.1° respectively for each. The process was repeated with the mean value being set at the identified optimal angular frequency for the window size, again, for each of the window sizes in the range (7 ≤ L ≤ 20). Hydrophobic moment plots for variable angular frequency were obtained for E(θ) = 100° for each window size in the range (7 ≤ L ≤ 21) residues and for the separate standard deviation values, σ = 0.1°, 0.3°, 0.5°, 0.7°, 0.9°, 1.1°. These were compared visually with the original plots obtained under the fixed angular frequency assumption (θ = 100°). In all cases, the bulk properties of the plots were similar irrespective of the level of dispersion introduced by the different values of the standard deviation. The hydrophobic moment plot for L = 15; θ = 100° is provided in figure 7. This is to be contrasted with the plots for L = 15; E(θ) = 100°, σ = 0.1°, σ = 0.7° and σ = 1.1°, also present in figure 7. Similar results were obtained for all other values, confirming, at least visually, that <μH> is robust to slight random perturbations about a fixed value. These properties were also observed for the simulation study with the fixed angular frequency assumption being violated about the optimum frequency for each of the window sizes in the range (7 ≤ L ≤ 20) and for each corresponding level of dispersion. Figure 7 Hydrophobic moment plot analysis of the transmembrane protein data set with varying standard deviation of θ about θ = 100°. Figure 7 shows hydrophobic moment plot analysis of the 403 transmembrane sequences of our data set using L = 15 and: In figure 7a, θ = 100°; in figure 7b, θ is from a Normal Distribution with E(θ) = 100° and standard deviation of 0.1° ; In figure 7c, θ is from a Normal Distribution with E(θ) = 100° and standard deviation of 0.7° and in figure 7d, θ is from a Normal Distribution with E(θ) = 100° and standard deviation of 1.1°. A more rigorous assessment of the variation was provided by analysis of the sample correlations. These were calculated in each case and compared to the empirically derived 99% confidence intervals established for window sizes in the range (7 ≤ L ≤ 20) under the fixed angular frequency assumption of θ = 100°. The calculated sample correlation coefficients were also compared to the point estimates for the original data. In all cases, the values were within the appropriate confidence intervals and were always close to the original sample correlation coefficient values, again providing evidence that <μH> is robust to random variation in angular frequency. The results of this investigation are given in table 3. Table 3 Sample correlation coefficients for optimum <μH> for θ = 100°, θ~N(100, σ2) and window sizes, L = 7, 11, 15, 16, 20. Window size (L) θ = 100; σ = 0 σ = 0.1 σ = 0.3 σ = 0.5 σ = 0.7 σ = 0.9 σ = 1.1 7 -0.576465 -0.576557 -0.576118 -0.574907 -0.577803 -0.575951 -0.577435 11 -0.476666 -0.476109 -0.475923 -0.476820 -0.476131 -0.475736 -0.475371 15 -0.312882 -0.312924 -0.312973 -0.313221 -0.313488 -0.312796 -0.311120 16 -0.180014 -0.180160 -0.180679 -0.180656 -0.179292 -0.178218 -0.180065 20 -0.156516 -0.156837 -0.156606 -0.156546 -0.158868 -0.158272 -0.155921 To test whether these correlations are artefactual, hydrophobic moment plots were obtained for the <μH> and <H> derived from the 403 artificial randomisation sequences generated by random re-ordering or randomisation [20] of each of the original optimum window sequences. The plot for a window size of L = 11 is given in figure 8. These analyses were undertaken for all those window sizes with previously identified statistically significant correlation coefficients between <μH> and <H> and were designed to test the importance of the spatial arrangement of the amino acids within the optimum sequences. Figure 8 Hydrophobic moment plot analysis of the transmembrane data set using randomised sequence arrangements. Figure 8 Hydrophobic moment plot analysis of our data set was performed using sequences generated by a) random rearrangement of sequences for the optimal windows, b) random sequences formed with amino acid relative frequencies the same as those of the optimal windows. In all cases, L = 11 and θ = 100°. Similar plots were obtained from Monte Carlo simulated data derived from the 403 sequences that had been generated by random sampling using the relative abundancies of residues found in the set of optimal windows. These analyses were therefore designed to look at the importance of relative amino acid composition for the correlations between <μH> and <H> and the results can be seen for a window size of L = 11 in figure 8. Again, analyses were performed for all window sizes with associated statistically significant correlations (data not shown). It is worth noting that since the effect of varying window size had a significant effect on the correlation between <μH> and <H>, varying L was observed to vary the optimal sequence identified within the transmembrane domain. Clearly this was not unexpected. Conclusions It can be seen from figure 5 that the most discriminating angular frequency for a fixed window size varies within the range, (95° ≤ θ ≤ 104°) for window sizes (7 ≤ L ≤ 20). There is an obvious damped oscillation present, which can be seen to correspond to the assumed intrinsic periodicity of α-helical secondary structure i.e. 3.6 residues per turn. Figure 5 clearly demonstrates that the fixed 100° angular frequency, assumed when modelling α-helices in general, is no more than a representative average with a value nearer 102° providing a maximum for an optimum L = 11 residue window in a transmembrane α-helical sequence. From figure 4, it is also evident that the degree of discrimination possible using <μH> declines in a near linear fashion with increasing window size with the optimum L = 11 residue window appearing to provide approximately average discrimination for transmembrane α-helices. The bootstrap derived 99% confidence intervals for the correlation coefficients between <μH> and <H> for window sizes in the range (7 ≤ L ≤ 20) showed that there are highly significant linear associations for the smaller window sizes in the range (7 ≤ L ≤ 16). As the magnitude of each of the corresponding sample coefficients is small (table 1), this should be interpreted as evidence of a strong (negative) association but with high variability being present. These correlations become weaker, on average, with increasing window size until they are not statistically significant at the 1% level and we have no compelling evidence that the variates are not independent. The choice of window size therefore, becomes paramount if <H> and <μH> are to be used to classify transmembrane α-helices. More importantly, the variation in correlation between these parameters and the effect of varying window size on the location of the sequence identified as optimal for α-helix classification brings into question the relevance of using the mean hydrophobic moment for comparison between varying window sizes. However, <μH> has been shown to be robust to departures from the fixed angular frequency assumption for a large range of window sizes appropriate for transmembrane proteins and for a range for levels of dispersion. There were no substantial differences between the plots for relative abundance sample data and those for the randomisation sequences (figure 8) except for a few chance negative <H> observations from the former. This suggests that there are no serial correlations between residue types, where presence in the identified section of the penetrating transmembrane stretch is determined predominantly by relative abundance. This is to be contrasted with the distribution of observations for the original transmembrane sequences for a window size of 11 residues (figure 1). Most noticeable is the difference in <μH> over the range of <H> values. There appears to be a lower bound for <μH> for the original sequence, which is clearly not present for the randomisation data. Furthermore, whilst the negative correlation would appear to be an artefact, as it is exhibited in all cases, the dispersion around any optimal fitted line through the data such as a least squares fit also is clearly different. It appears similar and quite spread out for the two randomised sequence data but considerably less so for the transmembrane sequences. This provides evidence that within the optimum window, whilst residue composition is not influential, order is. It would appear that this ordering is leading to both organisation and biological function for at least segments of the interacting portions of transmembrane proteins. This is consistent with the belief that the hydrophobic moment is a good predictor of amphiphilicity [8] although it can be unduly influenced by relatively few amino acid residues within a sequence [21]. In summary, our analyses confirm previous studies, which have shown limitations to the ability of hydrophobic moment plot methodology to assign function to membrane interactive α-helices [6]. More importantly, our investigation leads to a questioning of the logic of comparing mean hydrophobic moments, in general, for transmembrane proteins. This is due to the effect of window size on both, the correlation of mean hydrophobic moment with mean hydrophobicity and the identified sensitivity of the optimum window. Comparisons of the hydrophobic moment are seemingly only meaningful for separate transmembrane proteins with identical window sizes. Despite these limitations, <μH> has been shown to be robust to departures from the fixed angular frequency assumption for transmembrane proteins. Given the severe lack of structural information for transmembrane proteins, the identification of transmembrane α-helices using hydrophobic moment based analyses, and other bioinformatic approaches, seems likely to continue for the foreseeable future. Nonetheless, the results of such analyses should only be taken as a guide, and where possible, obtaining corroborative experimental data is essential. On the positive side, our results have demonstrated the importance of amino acid residue sequence order in establishing organisation and biological function for the transmembrane α-helices of proteins. With the ongoing development of predictive techniques, these results should be useful in furthering this development and helping to improve drug target identification. Methods The selection of transmembrane, α-helix forming segments The primary structures of 96 transmembrane proteins were selected from the Swiss-Prot data bank (; accessed 25.05.04) and confirmed as transmembrane by extensive analysis of the literature. The sequences were analysed for homology using the sequence alignment program BLAST (Basic local alignment search tool) [13] and twelve homologous sequences were rejected. From the remaining 84 primary structures, a data set comprising 403 putative transmembrane α-helical sequences, each of 21 residues, was established using the algorithm, Top Pred2 ([14]; ; accessed 25.05.04). Hydrophobic moment plot analysis of transmembrane, α-helix forming segments In the present study, all hydrophobic moment plot analyses were performed using the consensus hydrophobic scale of Eisenberg [4,5]. To identify putative transmembrane α-helix forming segments using hydrophobic moment plot methodology, hydropathy plot analysis [15] is initially undertaken to identify the primary amphiphilicity of candidate sequences. These sequences are selected using a 21 residue window as this is sufficiently long for an α-helix to span the bilayer. Once a putative transmembrane domain has been identified, an eleven residue window is considered to progress along the amino acid sequence and for each window, the hydrophobic moment at 100° is calculated. Based on the assumption that a protein sequence will adopt its most amphiphilic arrangement, the window with the maximum mean hydrophobic moment, <μH>, is taken as the most likely to form an amphiphilic α-helix [5]. The location of the optimum window was observed accordingly for window sizes of seven through to twenty consecutive residues. Optimal angular frequency and window length for <μH> For window sizes ranging from 7 to 20 amino acid residues <μH> were computed for the range of angular frequency values (95° ≤ θ ≤ 108°). In each case, the value of θ, which maximises <μH>, i.e. the value of θ which produces <μH>, was determined and is referred to as the optimal angular frequency for that window size. These procedures were based on previously published work, which identified variations in θ for α-helices [16]. Hydrophobic Correlation For window sizes ranging from 7 to 20 amino acid residues, scatterplots of <μH> versus <H> (hydrophobic moment plots) with θ = 100° were obtained. The corresponding sample correlation coefficients were calculated to identify the effect of window size on the relationship between these variates and hence on their ability to act as discriminators in the prediction of transmembrane α-helices. In addition, for each window size in the range (7 ≤ L ≤ 20) residues and for θ in the range (95° ≤ θ ≤ 108°), the response surface diagram for <μH> was constructed. Confidence intervals for the Correlation Coefficient Statistical confidence intervals were established for the Pearson (Product-Moment) Correlation Coefficient between <μH> and <H> for both cases where window size was varied for a fixed value of the angular frequency, and the angular frequency was varied for a fixed window size. The resulting mean hydrophobicity measures were checked for bivariate Normality and non-parametric bootstrap procedures [11] were used to estimate confidence intervals for the Correlation Coefficients [17]. To provide evidence of the statistical significance of any linear association, the bootstrap bias-corrected and accelerated technique (BCa) [18] and an analytical extension of this, the ABC [19]. In addition, the bootstrap Delta method was employed, which although another bootstrap based method, was developed specifically for estimating the variance of a function of sample means. As the sample Correlation Coefficient can be readily expressed as such a statistic, it is also well suited to the estimation of confidence intervals for these Correlation Coefficients [12]. As both main approaches differ substantially, a more informed assessment of statistical significance could therefore be made. Variable angular frequencies To assess the robustness of <μH> to the fix angular frequency assumption, e.g., θ = 100°, θ was varied randomly about 100° and <μH> was calculated for each of the optimal windows for window sizes (7 ≤ L ≤ 20) for the 403 transmembrane proteins. These calculations were also obtained for similar random variations about the observed optimum angular frequencies, again, for the various window sizes (7 ≤ L ≤ 20). In all cases, it is assumed that the variation follows a Normal distribution with the mean value set at the desired value for θ and with the standard deviation, σ, set at: 0.1°, 0.3°, 0.5°, 0.7°, 0.9° and 1.1° respectively for six separate Monte Carlo simulation studies. The sample correlation coefficients for each simulation were calculated and compared to the empirically derived 99% confidence intervals for the corresponding population values and, in particular, with the point estimates for the original sequences. Causality and biological function Given that these data are from an observational study, it is necessary to assess whether any linear associations between <μH> and <H> for the α-helix forming sequences of our data set are likely to be causal or merely an artefact of amino acid composition. To investigate these possibilities, two additional simulation studies were undertaken. The first looked at spatial arrangements of residues within the primary sequences and the second focused on the effect of amino acid composition on correlations between <μH> and <H>. To assess if positional or sequential correlational properties existed for the amino acids within the sequences, the sequence of residues for each of the optimum windows was re-ordered randomly. Artificial sequences were thus generated by random rearrangement or randomisation [20] of the primary sequences within the 403 optimal windows. Hence, each window associated with <μH> was used to generate a random arrangement. To further investigate whether correlations between <μH> and <H> were dependent on sequence composition and not on spatial or sequential correlation, an additional parametric bootstrap simulation study was conducted. Here 403 artificial sequences were created. Each was randomly generated where, for each position, selection was based on the relative abundance of all the residues for the complete 403 optimum windows. In both cases the corresponding <μH> and <H> from these newly created sequences were calculated, the associated hydrophobic moment plots obtained and sample correlations calculated. These were inspected to assess whether any linear associations for the original transmembrane data were thus likely to be causal or merely artefactual and whether, from inspection of variation, there was evidence of increased organisation, which could be interpreted as an indication of biological function. Supplementary Material Additional File 1 Transmembrane sequence data set Click here for file ==== Refs Brady AE Limbird LE G protein-coupled receptor interacting proteins: Emerging roles in localization and signal transduction Cellular Signalling 2002 14 297 309 11858937 10.1016/S0898-6568(01)00239-X Müller G Towards 3D structures of G protein-coupled receptors: a multidisciplinary approach Curr Med Chem 2000 7 861 888 10911020 Phoenix DA Harris F Daman OA Wallace J The prediction of amphiphilic α-helices Curr Protein Pept Sci 2002 3 201 221 12188904 Eisenberg D Weiss RM Terwilliger TC The helical hydrophobic moment: a measure of the amphiphilicity of a helix Nature 1982 299 371 374 7110359 Eisenberg D Schwarz E Komaromy M Wall R Analysis of membrane and surface protein sequences with the hydrophobic moment plot J Mol Biol 1984 179 125 142 6502707 Phoenix DA Stanworth A Harris F The hydrophobic moment plot and its efficacy in the prediction and classification of membrane interactive proteins and peptides Membr Cell Biol 1998 12 101 110 9829263 Auger IE Epand RM Computational techniques to predict amphipathic helical segments The Amphipathic Helix 1993 CRC Press, Florida, USA 7 19 Phoenix DA Harris F The hydrophobic moment and its use in the classification of amphiphilic structures (Review) Mol Membr Biol 2002 19 1 10 11989818 10.1080/09687680110103631 Harris F Wallace J Phoenix DA Use of hydrophobic moment plot methodology to aid the identification of oblique orientated α-helices Mol Membr Biol 2000 17 201 207 11302373 10.1080/09687680010018826 Phoenix DA Harris F Is use of the hydrophobic moment a sound basis for predicting the structure-function relationships of membrane interactive α-helices? Curr Protein Pept Sci 2003 4 357 366 14529529 Efron B Tibshirani RJ An Introduction to the Bootstrap 1993 Chapman & Hall, New York Efron B The Jackknife, the Bootstrap, and other Resampling Plans Regional Conference Series in Applied Maths 38, SIAM, Philadelphia 1982 Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389 Von Heinje G Membrane-protein structure prediction-hydrophobicity analysis and the positive-inside rule J Mol Biol 1992 225 487 494 1593632 Kyte J Dolittle RF A simple method for displaying the hydropathic character of a protein J Mol Biol 1982 157 105 132 7108955 Cornette JL Cease KB Margelit H Spouge JL Berzofsky JA De Lisi CD Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins J Mol Biol 1987 195 659 685 3656427 Sprent P Data driven Statistical Methods 1998 Chapman and Hall, London Buckland ST Monte Carlo methods for confidence interval estimation using the bootstrap technique Bull Appl Statist 1983 10 194 212 Efron B DiCiccio T More accurate confidence intervals in exponential families Biometrika 1992 79 231 245 Manly BFJ Randomization and Monte Carlo Methods in Biology 1991 Chapman and Hall, London Pewsey AR Phoenix DA Roberts MG Monte Carlo analysis of potential C-terminal membrane interactive α-helices Prot Pep Lett 1996 3 185 192

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==== Front BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-4-331532445810.1186/1471-2180-4-33Research ArticleFlagellin acting via TLR5 is the major activator of key signaling pathways leading to NF-κB and proinflammatory gene program activation in intestinal epithelial cells Tallant Thomas [email protected] Amitabha [email protected] Niladri [email protected] Joseph [email protected] Veer Michael J [email protected] Joseph A [email protected] Deparment of Cancer Biology, The Lerner Research Institute at the Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA2 Amitabha Deb, Massachusetts Biologics Labs, University of Massachusetts Medical School, 305 South Street, Jamaica Plain, MA 02130-3597, USA3 Michael de Veer, Centre for Animal Biotech, Department of Veterinary Science, Melbourne University, Parkville, Victoria, 3010, Australia2004 23 8 2004 4 33 33 6 5 2004 23 8 2004 Copyright © 2004 Tallant et al; licensee BioMed Central Ltd.2004Tallant et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Infection of intestinal epithelial cells by pathogenic Salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. The transcription factor NF-κB is a key regulator/activator of this gene program and is potently activated. We explored the mechanism by which Salmonella activates NF-κB during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to NF-κB activation in all the cells; invasion of cells by the bacteria is not required to activate NF-κB. Results Purified flagellin activated the mitogen activated protein kinase (MAPK), stress-activated protein kinase (SAPK) and Ikappa B kinase (IKK) signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by Salmonella. Flagellin expression was required for Salmonella invasion of host cells and it activated NF-κB via toll-like receptor 5 (TLR5). Surprisingly, a number of cell lines found to be unresponsive to flagellin express TLR5 and expression of exogenous TLR5 in these cells induces NF-κB activity in response to flagellin challenge although not robustly. Conversely, overexpression of dominant-negative TLR5 alleles only partially blocks NF-κB activation by flagellin. These observations are consistent with the possibility of either a very stable TLR5 signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both. Conclusion These collective results provide the evidence that flagellin acts as the main determinant of Salmonella mediated NF-κB and proinflammatory signaling and gene activation by this flagellated pathogen. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Lastly, TLR5 expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition. ==== Body Background Intestinal epithelial cells serve as a barrier between the luminal microflora and the body and as such are perfectly positioned to monitor the approach/invasion of pathogens. These intestinal epithelial cells (IECs) serve as innate immune sentinels and monitor their environment and constantly give out innate host defense instruction to local immune effector cells [1,2]. Pathogens such as Salmonella and other enteroinvasive pathogenic bacteria such as enteroinvasive E. Coli, Shigella and Yersinia upon infection of IECs leads to the up-regulation of the expression of host genes, the products of which activate mucosal inflammatory and immune responses and alter epithelial cell functions [3-6]. Previously we and others demonstrated that IKK via NF-κB and the SAPK signaling pathways via Jun N-terminal kinase (JNK) and p38 kinase were key regulators of the up-regulation of the proinflammatory gene program [3,7-9], with NF-κB appearing to be the most critical [3]. Typically Salmonella infects thirty-forty percent of IECs in culture models of infection [10], however, we and others have found that Salmonella infection activates NF-κB DNA binding activity to levels equivalent to that of TNFα which activates NF-κB in all of the cells [3]. Previous studies examining NF-κB activation by Salmonella in HT29 colonic intestinal epithelial cells, which serve as model colonic epithelial cells in culture, indicated that delivery of Salmonella proteins into the host cell via its type III secretion system (TTSS), such as SopE and SopE2, the bacterially encoded exchange factors for the Rho-family members Rac1 and CdC42, result in exchange factor activation, cytoskeletal rearrangements and activation of the MAPK, SAPK and NF-κB signaling pathways [7,8,11-15]. Recent observations that utilized Salmonella strains that were defective in invasion and delivery of invasion proteins by the TTSS but not attachment indicated that additional factors other than those delivered by the TTSS could lead to NF-κB activation [16]. Presently it is not clear what protein(s) dictate the activation of key signaling pathways that lead to the temporal expression of the proinflammatory gene program, although the SopE proteins have been given extreme attention recently [7,8,15]. In searching for additional Salmonella proteins that could activate the proinflammatory gene expression program, bacterial flagellin was recently found to be such a protein [16-19] and had been shown previously to activate IL-8 expression in monocytes [19-21]. Flagellin was found to activate NF-κB in polarized epithelial cells only when flagellin was present on their basolateral surface [22] consistent with the idea that a cell surface receptor was present there and could recognize it. The toll-like receptors (TLRs) have been found to recognize pathogen associated molecular patterns (PAMPs) reviewed in [23-26]. TLR2 interacts with TLR1 and TLR6 to recognize bacterial lipopeptides and zymosan respectively [27,28]. TLR4 recognizes LPS only when associated with its co-receptor MD2 and CD14 [29-32]. Recently, flagellin was demonstrated to be recognized by TLR5 and activate an innate host response [22,33]. However, little was known or demonstrated about the endogenous levels of TLR5 in cells used in those studies and why those cells failed to respond to flagellin. Here we have identified flagellin as the primary initiator and temporal regulator of not only the major signaling pathways activated during Salmonella infection but also of key target genes of the proinflammatory gene program too. We have also found flagellin expression to be required for Salmonella bacterial invasion. Independently we found TLR5 recognizes flagellin but its signaling activity toward this PAMP is consistent with either the aid of another flagellin-recognizing co-receptor (as TLR4 utilizes for LPS) or the use of another adapter protein, perhaps similar to MyD88, that is absent or present at low levels in flagellin non-or low-responding cells. Results Salmonella infection leads to a minority of cells invaded but activates NF-κB in nearly all cells Previously, we have noted that pathogenic Salmonella sp. infection leads to potent IKK and NF-κB activation and activation of the proinflammatory gene program [3]. Previous studies suggest that about thirty-forty percent of the intestinal epithelial cells are infected during a typical Salmonella infection in cultured intestinal epithelial cells [10]. We wished to address the question of how bacterial infection of about thirty percent of the host cells could give rise to NF-κB DNA binding activity equivalent to activation of NF-κB in nearly all of the host cells as TNFα treatment of the cells does. To examine this phenomenon in detail HT29 cells either mock-infected or infected at a MOI of fifty for one-hour with wild-type S. typhimurium that had been transformed with the plasmid pFM10.1, that encodes green fluorescent protein (GFP) under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell [34]. As can be seen in Fig. 1A, GFP expression occurs in about thirty to forty percent of the cells. We next examined the localization of the NF-κB subunit p65 (RelA) in non-treated (mock-infected), Salmonella infected or TNFα (10 ng/ml) stimulated cells and found that p65 (RelA) was localized to the cytoplasm in non-treated cells whereas, in Salmonella infected cells or in TNFα treated cells p65 (RelA) had localized to the nucleus (Fig. 1B). These results demonstrate that Salmonella infection activates NF-κB in virtually all of the cells even though only a minority of them become infected and is consistent with and aids in explanation of our previous results examining Salmonella infection and NF-κB activation [3]. Figure 1 Salmonella infection leads to NF-κB nuclear localization even in non-infected cells. HT29 cells were grown on glass coverslips and either mock-infected, left untreated, infected with Salmonella typhimurium, or treated with TNFα (10 ng/ml). Cells fixed after 30 min (TNF) and 1 h (Salmonella) as described in Experimental Procedures and Salmonella that had invaded HT29 cells were detected by direct fluorescence microscopy of GFP expression, p65(RelA) localization was monitored by indirect immunoflourescence of rabbit anti-p65 antibody detected with FITC-conjugated donkey anti-rabbit antibody. DAPI was used to stain nuclei. A, HT29 cells were mock-infected or infected at an MOI of 50 with Salmonella typhimurium strain SJW1103G which expresses GFP from the ssaH promoter that is only active inside infected host cells [10,34]. Cells were photographed using bright field microscopy (BF), and immunoflourescence to detect GFP or DAPI staining as indicated. Images were merged (overlay) to reveal cells that were infected. B, HT29 cells were left untreated, infected with Salmonella typhimurium strain 1103 or treated with TNFα. NF-κB p65(RelA) localization under various conditions as indicated was monitored by indirect immunofluorescence. Cells were visualized by bright field microscopy (BF), cell nuclei were stained with DAPI and p65(RelA) was visualized with FITC. DAPI staining was falsely colored red to make visualization of the merge (overlay) easier to distinguish. Soluble bacterial product identified as flagellin can activate NF-κB in intestinal epithelial cells Since Salmonella sp. infection of intestinal epithelial cells in culture led to only roughly thirty percent infection but activation of NF-κB in nearly all of the cells, we anticipated that NF-κB activation was in response to host cell recognition of bacteria structural components or products produced by the bacteria and not by the invasion process. Invasion itself has been demonstrated not to be required for activation of the proinflamatory gene program as had previously been thought [16]. To investigate this possibility sterile-filtered S. dublin culture broth left either untreated or boiled for twenty minutes was used to challenge HT29 intestinal epithelial cells and NF-κB DNA binding activity was monitored by electromobility shift assays (EMSAs) of whole cell extracts (WCE) prepared forty-five minutes after exposure [3,35]. Potent activation of NF-κB in response to the broth under both conditions was observed indicating the activating factor was heat-stable (AD, TT and JD, personal observations) and is not LPS since HT29 cells are not responsive to LPS [3,35]. The native sterile-filtered concentrated broth was subsequently treated with DNase, RNase, proteinase K or crudely size fractionated on 100 kDa centricon filters. The variously treated broths were then used to challenge HT29 intestinal epithelial cells and WCEs were prepared after forty-five minutes and NF-κB DNA binding activity was analyzed by EMSA (Fig 2A). Direct infection of HT29 cells by S. typhimurium 1103 or exposure to the culture broths (supt), as indicated, induced NF-κB DNA binding activity, while the activity-inducing factor was found to be sensitive to protease digestion and was retained by a 100 kDa filter (Fig. 2A). To further determine the identity of the NF-κB inducing activity, sterile-filtered concentrated broth culture was fractionated by Superose 12 gel permeation chromatography (Fig. 2B) and by anion exchange chromatography (Fig. 2C). Aliquots of chromatography fractions were assayed for their ability to activate NF-κB in HT29 cells and analyzed by EMSA. As can be seen from the Coomassie blue stained gel (Fig. 2B, top panel) increased NF-κB DNA binding activity (Fig. 2B, lower panel lanes 4–6) corresponded to the increased abundance of an approximately 55 kDa protein. Anion exchange chromatography on POROS HQ matrix and elution of bound proteins with an increasing salt gradient as indicated (Fig. 2C) demonstrated that NF-κB DNA binding-inducing activity corresponded to chromatographic fractions containing an increased abundance of the 55 kDa protein (Fig. 2C top panel, and data not shown). Eluted fractions observed in Fig. 2C were concentrated and fractionated on preparative 12% SDS-PAGE gels and bands corresponding to B1-B6 were cut from the gels and the proteins eluted, precipitated and renatured as described in Experimental Procedures and used to stimulate HT29 cells. Whole cell extracts from these cells were assayed for NF-κB DNA binding-inducing activity by EMSA and only band 2 (B2) corresponding to the 55 kDa protein (Fig. 2C lower panel) was able to elicit NF-κB DNA binding activity while buffer from the beginning or end of the salt gradient failed to activate NF-κB DNA binding activity. Figure 2 Protein factor in Salmonella culture broth leads to NF-κB activation. A, Salmonella dublin culture broth concentrated 100-fold was treated as indicated or infectious bacteria, as indicated was used to challenge HT29 cells. NF-κB DNA binding activity was assayed by EMSA from whole cell extracts prepared 45 min after treatment. Authenticity of the NF-κB DNA:protein complex was determined using p65(RelA)-specific and p50-specific antibody supershifts. B, Concentrated Salmonella dublin culture broth (IN) was chromatographed by gel permeation on a Superose 12 column. Eluted protein fractions were analyzed by fractionation on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Molecular weight markers for chromatography and on the gels are indicated. Aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. C, Concentrated Salmonella dublin culture broth (IN) was chromatographed by anion exchange chromatography on POROS HQ matrix. Proteins were eluted with an increasing NaCl gradient as indicated and analyzed on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Input and aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. Eluted material corresponding to protein bands B1-B6, a blank portion of the gel was isolated from a duplicate 10% SDS-PAGE gel as described in Experimental Procedures along with buffer samples from the beginning and end NaCl buffer gradient and used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. Proteins corresponding to protein bands B1-B6 and blank areas of the gel were further processed for peptide sequencing as described in Experimental Procedures. Trypsin digestion of the protein corresponding to B2 and analysis by electrospray ion trap LC/MS identified the amino acid sequence of twenty-one peptides. Flagellin (seventy-five percent coverage by the twenty-one peptides) was unambiguously identified as the protein consistent with inducing NF-κB DNA binding activity (Fig 3). Figure 3 Identifcation by mass spectrometry of flagellin as the NF-κB activating factor in Salmonella culture broth. Microcapillary HPLC tandem mass spectrometry of Band 2 digested by trypsin. Peaks corresponding to Salmonella peptides are numbered and identified with the corresponding numbered peptide sequence to the right. Flagellin is required to activate NF-κB in intestinal epithelial cells To determine if flagellin was indeed the factor that was responsible for triggering activation of NF-κB after exposure of intestinal epithelial cells to direct bacterial infection or to filtered culture broths of pathogenic Salmonella sp. we prepared infectious bacteria and boiled and filtered culture broths from the non-flagellated E. Coli DH5α, pathogenic S. dublin strain 2229, an isogenic S. dublin 2229 SopE- mutant, isogenic S. dublin 2229 SopB- mutant, isogenic S. dublin 2229 double SopE-/SopB- mutant (strain SE1SB2), S. typhimurium strain 1103, and isogenic S. typhimurium fliC::Tn10 insertion mutant (strain 86) and a S. typhimurium 1103 isogenic double mutant fliC-/fljB- and were used to challenge HT29 cells. Bacteria and culture broths were used to challenge HT29 intestinal epithelial cells and WCE extracts were prepared after forty-five minutes and analyzed for NF-κB DNA binding activity by EMSA. Salmonella strains could activate NF-κB, while Salmonella strains failing to produce flagellin (fliC and fliC-/fljB- mutants as indicated) also failed to activate NF-κB (Fig. 4A &4B). E. Coli DH5α is non-flagellated and does not produce flagellin failed to activate NF-κB. We also noticed through numerous experiments that S. dublin direct infections always activated NF-κB to a greater extent than S. typhimurium as observed in Fig. 4A while culture broths from both species activated NF-κB almost equally well (Fig. 4B). We believe this difference is due perhaps to S. dublin releasing more flagellin into the cell culture media than S. typhimurium during infection since purification of flagellin from both S. dublin and S. typhimurium and addition of equivalent amounts of chromatographically purified flagellin gave similar NF-κB activation profiles (TT & JD, unpublished observations). Figure 4 Flagellin mutants fail to activate NF-κB. EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min from non-infected cells (UN) and after direct infection of HT29 cells with wild-type E. coli DH5α, wild-type Salmonella dublin or SopE- mutant, SopB- mutant, the SopE-/SopB- double mutant, wild-type Salmonella typhimurium strain 1103, the fliC- mutant (fliC::Tn10), the fliC-/fljB- double mutant as indicated at an MOI of 50. B, EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min after challenge of HT29 cells from non-infected cells (UN) or with sterile-filtered concentrated culture broths from wild-type and mutant bacteria as indicated. Of note is the total failure of the double flagellin gene mutants to activate NF-κB as compared to the very minor activation observed in the single Phase I flagellin fliC::Tn10 insertion mutant (next to last lanes in Fig. 4A &4B) which likely is due to the extremely limited expression of the phase II flagellin (from fljB), although the strains of Salmonella used here genetically are unable or rarely shift phases of flagellin production. These results are consistent with previous reports identifying flagellin as a potent inducer of the proinflammatory response and IL-8 production [16-19]. Since flagellin appears required for activation of the NF-κB pathway upon direct infection of intestinal epithelial cells it appeared possible that flagellin may also be the major determinant of other major mitogenic and stress activated signaling pathways activated upon pathogenic Salmonella infection of intestinal epithelial cells. Previously others and we have demonstrated that direct Salmonella infection of intestinal epithelial cells results in JNK activation [8] and also the activation of NF-κB via IKK [3]. The identification of flagellin as a potent NF-κB activator is significant since SopE had previously been shown to be a pathogenic Salmonella bacteriophage encoded protein that is injected into the host cell and acts as an exchange factor for the small Rho GTPases Rac1 and CdC42 initiating cytoskeleton rearrangements and eventual activation of the MAPK, SAPK and NF-κB pathways [7,15], while SopB is a Salmonella protein that functions as an inositol phosphate phosphatase and participates in cytoskeletal rearrangements and stimulates host cell chloride secretion [36]. Flagellin triggers activation of the mitogen activated protein kinase, stress activated protein kinase and IKK signaling pathways Intestinal epithelial cells act as sentinels for invasion of luminal surfaces and orchestrate the attraction of effector immune cells to the area by production of chemokine genes like IL-8 and macrophage chemoattractant protein 1 (MCP1) proinflammatory cytokine genes such as TNFα, IL-1 and IL-6 [1,4-6]. Expression of these genes primarily depends upon the action of transcription factors that are activated in response to the transmission of signals via the MAPK, SAPK and IKK signaling pathways. Since NF-κB is considered a central regulator/activator of the proinflammatory gene program we decided to examine the effect that non-flagellin producing mutant strains of Salmonella had on activation of the MAPK, SAPK and IKK signaling pathways compared to infection of intestinal epithelial cells with wild-type Salmonella or by exposure of the intestinal epithelial cells to purified flagellin. Infection of HT29 cells with wild-type S. typhimurium resulted in activation of MAPKs ERK1&2, the SAPKs p38, JNK and IKK (Fig. 5) as determined by use of activation-indicating phospho-specific antibodies in immunoblot (IB) analysis or antibody-specific immuno-kinase assays (KA) for JNK and IKK using their respective substrates GST-cJun 1–79 and GST-IκBα1–54 [37-39]. Interestingly, MAPK stimulation is transient in nature as activation declines beginning at forty-five minutes while p38, JNK and IKK activity increases with time through one hour. As seen in Fig. 4, the fliC-/fljB- double mutant Salmonella also failed to induce IKK and NF-κB activity (Fig. 5 as indicated). Surprisingly, the fliC-/fljB- double mutant Salmonella failed to induce the SAPKs p38 and JNK and only briefly (fifteen minutes) activated MAPK. This result is puzzling since other Salmonella proteins such as SopE and SopE2 can activate the small GTPases Rac and CdC42, and these Rho family GTPases have been linked to JNK and p38 activation [7,8,14,15] yet appear not to function in the flagellin minus strain. Figure 5 Flagellin is required for activating multiple signaling pathways during Salmonella infection and leads to nuclear localization of NF-κB. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 15 min, or infected with either wild-type (WT) Salmonella typhimurium strain 1103 or the Salmonella typhimurium double fliC-/fljB- mutant strain 134 as indicated. WCE were prepared at the indicated times or at 10 min for TNF-treated cells or 15 min for anisomycin/PMA treated cells and used in EMSAs to analyze NF-κB DNA binding activity, or in immuno-kinase assays (KA) using anti-IKK or anti-JNK antibodies to measure IKK and JNK kinase activity on their respective substrates GST-IκBα 1–54 and GST-cJun 1–79 (as indicated). Immunoblot (IB) analysis of equivalent amounts (40 μg) of protein from each extract was fractionated on SDS-PAGE gels and transferred to PVDF membranes and probed with the indicated antibodies to detect bulk IKK, JNK, ERK and p38 as indicated. Immunoblot analysis using phospho-specific antibodies for ERK and p38 to detect activated ERK and p38 are indicated. B, Immunofluorescence demonstrating that flagellin mutant Salmonella fail to infect HT29 cells and that purified flagellin stimulation of HT29 cells leads to NF-κB nuclear p65 (RelA) localization as determined by indirect immunofluorescence. Imaging of the treatment indicated HT29 cells grown on coverslips was essentially the same as in Fig. 1A & 1B. False coloring of the DAPI stain was used to enhance the visualization of both DAPI stained nuclei and p65 nuclear localization. The fliC-/fljB- double mutant Salmonella failed to invade HT29 cells compared to the wild-type Salmonella strain as determined by gentamycin protection/invasion assay (see Experimental Procedures). The flagellin fliC-/fljB- double mutant displayed a four orders of magnitude difference in its ability to invade HT29 cells (TT & JD, unpublished observations). To demonstrate this point further, we infected HT29 cells with either wild-type Salmonella or the fliC-/fljB- double mutant Salmonella (strain 134), both strains were transformed with the plasmid pFM10.1 that encodes GFP under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell [10,34]. The wild-type Salmonella clearly was able to infect HT29 cells (GFP, Fig. 5B) while the flagellin mutant bacteria failed to invade HT29 cells as evidenced by the lack of GFP expression (Fig. 5B). To determine if flagellin is sufficient or that other bacterially produced proteins are required for invasion, we added either purified flagellin or sterile-filtered culture broths or a combination of both to HT29 cells that were challenged with the Salmonella fliC-/fljB- double mutant and assayed for invasion. Intestinal epithelial cells failed to be invaded using all tested combinations of purified flagellin and/or culture broths with the fliC-/fljB- double mutant strain (TT & JD, unpublished observations). To our knowledge there is no known direct connection between expression of flagellin genes and the effectiveness of the type III secretion system to deliver bacterially produced proteins such as SopE, SopE2 and SipA or other Sip or Sop proteins [7,14,15,40,41] that play important roles in initiating bacterial internalization. Furthermore, to evaluate the effectiveness of flagellin to stimulate p65 (RelA) nuclear localization in intestinal epithelial cells we challenged HT29 cells with purified flagellin and examined p65 (RelA) localization using indirect immunofluorescence and found p65 (RelA) nuclear localization in nearly every cell (Fig. 5B as indicated). Purified flagellin (0.5 μg/ml) was added to the culture media of HT29 cells and WCE were prepared at various times as indicated after exposure and assayed for NF-κB DNA binding activity in EMSAs (Fig. 6A). Flagellin potently activated NF-κB in a time dependent manner similar to that observed for TNF (10 ng/ml) treatment of HT29 cells (Fig. 6A). Analysis of the MAPK, SAPK and IKK signaling pathways (Fig. 6B) at various times after flagellin treatment of HT29 cells using activation-specific phospho-antibodies to monitor MAPK and p38 kinase activation or antibody-specific immunoprecipitation kinase assays for JNK and IKK activities demonstrated that JNK and IKK activity increased through time to one-hour while p38 and MAPK (ERK1&2) activity peaked at thirty minutes and began to decline to noticeably lower levels by one-hour (Fig 6B as indicated). The activation profile of the MAPK, SAPK and IKK signaling molecules ERK1&2, p38, JNK and IKK in intestinal epithelial cells in response to purified flagellin exposure remarkably resembled that of intestinal epithelial cells infected with wild-type Salmonella (Fig. 5A). From these observations we conclude that the temporal activation of the signaling pathways examined here (MAPK, SAPK and IKK), which reflect early events in Salmonella infection, are determined almost exclusively by recognition and response of intestinal epithelial cells to flagellin. Figure 6 Purified flagellin activates signaling pathways and proinflammatory gene expression in intestinal epithelial cells mimicking that of wildtype a wild-type Salmonella infection. HT29 cells were left untreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin [An] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 μg/ml) for the indicated times. WCE were prepared and analyzed by EMSA for NF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblot analysis using phospho-specific antibodies for ERK or p38 to detect activation and with kinase-specific antibodies as described in Fig. 5A to detect bulk kinase abundance as indicated. A, EMSA to detect NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (α p65), normal rabbit serum (NRS) served as an irrelevant antibody control. B, immunoblot and kinase assays to detect IKK, JNK, ERK and p38 kinase activities and protein abundance as in Fig. 5A. C, semi-quantitative RT-PCR of proinflammatory gene expression of non-treated, wild-type and flagellin double mutant Salmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 μg/ml) stimulated cells. HT29 cells were harvested at the indicated times after the indicated treatments and isolated RNA was used to make first strand cDNA that subsequently used in RT-PCR reactions (as described in Experimental Procedures) using gene-specific primers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was used as a standard for normalizing expression patterns. Resulting PCR products were fractionated on 2% agarose gels and visualized by eithidium bromide staining. We wished to further examine the effect of purified flagellin and flagellin present on Salmonella on the temporal pattern of proinflammatory cytokine gene expression in intestinal epithelial cells in order to differentiate the effects of flagellin alone vs. flagellated Salmonella or non-flagellated Salmonella infection. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml), or stimulated with flagellin (0.5 ug/ml), or infected with wild-type Salmonella typhimurium or the Salmonella fliC/fljB double mutant (at MOI of 50). After the indicated times after treatment or infection, HT29 cells were harvested in ice-cold PBS and the cell pellets lysed in Trizol and RNA was purified and used to prepare first-strand cDNA (see Experimental Procedures). Aliquots of the cDNA were used in semi-quantitative RT-PCR reactions using IL1α, IL-1β, IL-8, TNFα, MCP1 and β-actin gene specific primers (sequences available upon request) and the products were fractionated on ethidium bromide containing 1.2% agarose gels. Expression of the known NF-κB target genes IL-1β, IL-8, TNFα and MCP1 was increased in response to TNFα or purified flagellin exposure (Fig. 6C). Wild-type Salmonella infection also led to activation of these same genes although the expression of TNFα and MCP1 was transient in comparison and occurred immediately after infection. The Salmonella fliC-/fljB- double mutant failed to induce IL-1β, IL-8 and TNFα expression, however MCP1 expression was induced, although at lower levels than that induced by wild-type Salmonella, and also, the expression of MCP1 was not transient in nature and continued throughout the time course (9 h) (Fig. 6C). The expression level of β-actin served as an internal standard for comparison. Interestingly, IL-1α, which is not an NF-κB target gene was stimulated in response to HT29 cell challenge by all of the treatments. Obviously, the Salmonella fliC-/fljB- double mutant can activate other signaling pathways leading to IL-1α expression. We presently do not know what these signaling pathways are. Flagellin activates NF-κB DNA binding in a MyD88-dependent manner Flagellin was capable of activating the requisite signaling pathways consistent with proinflammatory gene activation similar to that of a cytokine like TNFα that activates all cells on which a functional cell surface receptor for it is present (see p65 [RelA] nuclear localization in Fig. 1 and Fig. 5C) we decided to examine the potential of the Toll-like receptors, to activate the NF-κB pathway in response to flagellin exposure. To quickly test this hypothesis we examined the effect that a dominant-negative MyD88 (aa 152–296) [42] expressing adenovirus had on flagellin-mediated NF-κB activation in HT29 cells. MyD88 is an adapter protein utilized by the IL-1 receptor and all of the known TLRs, which share homology to IL-1 through their cytoplasmic signaling domain and is required for immediate activation of the NF-κB pathway [43,44]. We found that expression of DN-MyD88 in HT29 cells blocked the activation of NF-κB DNA binding activity assayed by EMSA analysis in response to IL-1 or flagellin exposure, consistent with the action of a TLR-mediated activation of NF-κB (TT & JD, unpublished observations). To examine this possibility further we initially used wild-type, MyD88-/- and TLR2-/-/TLR4-/- MEFs (a gift of S. Akira, Univ. of Osaka, JA) to verify the role of MyD88 and to examine the potential role of two of the TLRs to respond to flagellin or to direct wild-type Salmonella infection and lead to NF-κB activation (Fig. 7). Wild-type Salmonella infection activates NF-κB potently in both the wild-type and TLR deficient MEFs (lanes 2 & 15) but this activation is somewhat defective in the MyD88 deficient MEFs (lane 10). Challenge of all three types of cells with concentrated sterile-filtered wild-type S. dublin or the double SopE-/SopB- isogenic mutant S. dublin strain SE1SB2 culture broths activated NF-κB in wild-type MEFs and TLR2/4 double deficient cells but failed to activate NF-κB in MyD88 deficient cells (compare lanes 11 and 12 with lanes 3, 4, 6, 7, 16 and 17). NF-κB was potently activated in wild-type MEFs by exposure to purified flagellin (0.5 μg/ml) and therefore eliminated the possibility that LPS played a role in NF-κB activation in these experiments. The exclusion of LPS as a major contributor to NF-κB activation is also provided by the potent activation of the TLR2/4 double deficient MEFs (lanes 16 & 17). TLRs 2 and 4 respond to bacterial lipopeptides, peptidoglycans, certain LPSs and gram negative LPS respectively [45-47]. IL-1 stimulation verified the functional requirement of MyD88 in transmission of IL-1 and flagellin-mediated signals. Figure 7 Flagellin-mediated activation of NF-κB is MyD88 dependent. Infectious wild-type Salmonella Dublin (MOI of 100), IL-1 (20 ng/ml), purified flagellin (1 μg/ml) (as indicated), sterile-filtered and concentrated 100 kDa filter retentate supernatant (spt) from wild-type Salmonella dublin and SopE-/SopB- double mutant Salmonella dublin strain SE1SB2 (S2, as indicated) was used to challenge wild-type, MyD88-/- knockout or TLR2-/-/TLR4-/- double knockout MEFs as indicated. WCEs were prepared 45 min after treatments and examined by EMSA to analyze NF-κB DNA binding activity. IL-1 (20 ng/ml) was used as a positive control to monitor MyD88 function. To further define a possible role for the TLRs in flagellin recognition we assayed for the ability of overexpressed TLRs to activate NF-κB in cells that normally respond poorly to flagellin exposure. Choosing cells that responded slightly to purified flagellin ensured that the signaling components and adapters that flagellin uses were present and functional and that the limiting factor was likely only to be the receptor that responds to flagellin. We found that HeLa cells and HEK293T cells activated NF-κB DNA binding activity in response to IL-1 stimulation but poorly to flagellin exposure (TT & JD, unpublished observations) (but see Fig. 9B) and we chose HEK293T cells to use further because of their greater transfection efficiency. Amino-terminus FLAG epitope-tagged TLRs 1–9 (kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) [48,49] were overexpressed in HEK 293T cells in transient transfections along with the 2×-NF-κB-dependent promoter driven luciferase reporter gene [50] and the expression of luciferase in response to no treatment, flagellin (0.5 μg/ml) or TNFα (10 ng/ml) was determined. TLR5 was the only TLR whose expression resulted in a noticeable response to flagellin challenge of the cells (Table 1). Figure 9 TLR5 is expressed in numerous cell types and has variable responses to flagellin. A, whole cell extracts were prepared from non-stimulated T84, HT29, A549, HeLa, 293T and T98G cells and fractionated on a 8% SDS-PAGE gel, proteins were transferred to PVDF membrane and probed with anti-TLR5 antibody for immunoblot analysis (IB). Protein loading was examined by probing with anti-actin antibody. B, HT29, A549, HeLa, 293T and T98G cells were left untreated (--), treated with flagellin (F) or TNFα (T) and WCEs were prepared after 45 min and used in EMSA to monitor NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (αp65), normal rabbit serum (NRS) served as an irrelevant antibody control. C, HT29, A549, HeLa, 293T and T98G cells WCEs (50 μg) were fractionated on a 8% SDS-PAGE gel, proteins transferred to Immobilon P and immunoprobed with anti-muc1 (1:450, Santa Cruz). Size markers are listed and muc1 position is indicated with an arrow. Table 1 TLR5 reponds to flagellin and activates NF-κB No Stim TNF Flic Vector 1 13.5 4.9 TLR1 1.7 ND 5.1 TLR2 1.6 ND 5.3 TLR3 1.5 ND 5.0 TLR4 1.8 ND 5.4 TLR5 1.6 ND 9.2* TLR7 1.5 ND 5.2 TLR8 1.4 ND 5.0 TLR9 1.5 ND 5.1 293T cells were transfected with empty vector (pCDNA3.1) or the individual listed wild-type TLR alleles in triplicate in 6-well dishes. Cells were left untreated (No Stim), TNFα (10 ng/ml) or flagellin (1 μg/ml). NF-κB reporter activity was adjusted by normalizing expression to control Renilla luciferase activity and fold induction was calculated as reporter gene activity in treated cells/reporter gene activity in non-stimulated cells. ND is not determined. To further determine the likelihood of TLR5 being the TLR through which flagellin activated NF-κB, we constructed dominant-negative signaling mutations by deletion of the carboxyl portion of each TLR to a conserved tryptophan in the TIR domain (see Materials and Methods). A similar mutation in the IL-1 receptor abrogates its ability to lead to NF-κB activation [51,52]. Each DN-TLR along with a reverse cloned TLR5 (AS-TLR5) were cloned into the mammalian expression vector pCDNA3.1 (Invitrogen, Carlsbad, CA). All mutant proteins were expressed well (TT & JD unpublished observations). Each DN-TLR mammalian expression vector and empty expression vector along with 2× NF-κB Luc was transfected as previously described [3] into HT29 cells which respond very well to flagellin. The transfected cells were left untreated, stimulated with TNFα (10 ng/ml) or with purified flagellin (0.5 μg/ml). Reporter gene expression was observed not to be affected by DN-TLR expression in response to TNFα stimulation of transfected cells (Fig. 8A) however, only expression of either the DN-TLR5 or an antisense TLR5 construct resulted in a nearly fifty percent and twenty-five percent inhibition of flagellin-mediated reporter gene activation respectively (Fig. 8B), while DN-TLR2 also was found to mildly inhibit flagellin-mediated reporter expression. These results imply that TLR5 takes part in cell surface recognition of flagellin and initiates the signaling pathway leading to NF-κB activation. The effect of DN-TLR2 on NF-κB-dependent reporter gene activation may be non-specific since its expression also inhibited TNFα-mediated reporter activation as compared to the other DN-TLRs. DN-TLR2 may also compete for an unknown adapter protein that both TLR2 and TLR5 might share. In any event, TLR2 and TLR4 were shown by the results presented in Fig. 7 not to be required for flagellin-mediated activation of NF-κB. Figure 8 TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown. Flagellin-mediated activation of NF-κB in intestinal epithelial cells leads to increased and decreased expression of a subset of TLRs Stimulation of intestinal epithelial cells with S. typhimurium or with purified flagellin led to activation of the proinflammatory gene program (Fig. 6C). We wished to examine whether or not expression of TLR genes would also be altered in flagellin stimulated cells. HT29 cells were treated or not with purified flagellin (0.5 μg/ml) or with TNFα (10 μg/ml) and total RNA was isolated from non-treated and treated cells three hours after stimulation and used to make first-strand cDNA. Real-time RT-PCR using gene-specific primers for each of the TLRs (Superarray, Frederick, MD) and first-strand cDNA prepared from non-stimulated or flagellin stimulated cells was used to generate SYBR-green (Perkin-Elmer) labeled DNA products that were detected in an iCycler™ (Bio-Rad). Interestingly, flagellin only mildly activated the expression of TLR2, while expression levels of TLRs 5, 6, 9 and 10 were decreased by 2-fold (Table 2). Contrastingly, TNF stimulation led to increased expression of TLRs 3 and 4 (1.6- and 3.5-fold respectively), while TLRs 2, 9 and 10 were decreased by approximately 2-, 5- and 3-fold respectively. GAPDH expression served as comparative standard. Table 2 Change in TLR mRNA levels following TNFα or FliC stimulation. Fold change Fold change Gene TNFα stimulated FliC stimulated TLR1 ND ND TLR2 0.6 1.3 TLR3 1.6 0.6 TLR4 3.5 1.1 TLR5 0.9 0.5 TLR6 0.9 0.6 TLR7 M M TLR8 ND ND TLR9 0.2 0.5 TLR10 0.3 0.5 GAPDH 1.0 1.0 ND None detected by RT2PCR M None detected above level of minus RT control. Reverse Transcription and Real Time PCR (RT2PCR)-RNA was prepared from cells left untreated, stimulated with TNFα (10 ng/ml) or flagellin (1 μg/ml) for 3 h. RT2PCR was performed with an iCycler (Bio-Rad) to quantify SYBR-green labeled products generated from PCR products of 1st strand cDNA prepared from TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT2PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 5 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT2PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Fold change in mRNA expression was expressed as 2ΔΔCt. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. TLR5 is expressed in cells that don't respond well to flagellin This study and others [22,33] have identified TLR5 as the likely TLR through which flagellin activates NF-κB. Previous reports made no determination on the presence or abundance of TLR5 in the cells that they used to ascertain its function [22,33]. We wished to determine if TLR5 protein abundance was absent or greatly decreased in cells that failed to respond or responded poorly to challenge by flagellin. TLR5 abundance in a number of cell lines was examined by immunoblot analysis using a TLR5-specific antibody and compared with the ability of purified flagellin to induce NF-κB DNA binding activity of WCEs prepared from them. Intestinal epithelial cell lines T84 and HT29 were used as was the lung adenocarcinoma cell line A549, the human cervical adenocarcinoma cell line HeLa, the human embryonic kidney cell line expressing large T antigen HEK293T, and the glioblastoma cell line T98G. TLR5 protein was detected in all cell lines examined by immunoblot with TLR5-specific antibody (Fig. 9A). T84 cells exhibited the highest abundance while expression levels of the other cell lines were similar and appeared not to differ by more than two-fold (Fig. 9A). NF-κB DNA binding activity in non-stimulated, TNFα and flagellin stimulated cells was analyzed by EMSA assays of WCEs prepared from each cell type (Fig 9B). HT29 and A549 cells responded strongly to flagellin and to TNFα stimulation while HeLa, 293T and T98G cells responded poorly (HeLa, 293T) or not at all (T98G) to flagellin stimulation. The authenticity of the NF-κB DNA binding complex was determined using p65-specific antibody to supershift the NF-κB DNA:protein complex. It is of interest that some cells that express TLR5 either do not respond at all or do so very poorly. This may be due to either lack of receptor presence at the plasma membrane and intracellular localization, inactivating or detrimental mutations in the TLR5 gene in these cell lines or lack of or low abundance of a required co-receptor or adapter protein (as is the case in some cells for TLR4 and its co-receptor/adapter MD2 [30,53,54]). IL-1 can activate NF-κB DNA binding activity in all of the examined cell lines so it appears that the signaling apparatus downstream of MyD88 to NF-κB is intact. Recently Muc1 a secreted and membrane bound mucin protein was shown to serve as a receptor that bound Pseudomonas aeruginosa and its flagellin, leading to activation of the MAPK pathway [55,56] although NF-κB activity was not examined. We examined the muc1 abundance levels in HT29 (strong flagellin responder), A549 (strong flagellin responder), HeLa, 293T (both weak flagellin responders) and T98G (no flagellin response) to determine if its expression correlated with the activation profile of NF-κB and MAPK in these cells in response to flagellin [55]. Should this be the case then muc1 might serve as a viable co-receptor for TLR5 in propagating activation signals leading to NF-κB activation. We observed that only HT29 cellular proteins gave a strong signal by immunoblot analysis using an muc1-specific antibody while muc1 was barely detectable in the other cell lines (Fig. 9C). These results suggest that muc1 does not serve the role of a TLR5 co-receptor that leads to NF-κB activation and likely plays little to no role activating MAPK pathways in A549 cells where we have observed similar temporal MAPK activation in response to flagellin exposure as we do in HT29 cells (TT and JD, unpublished results). Further examination of muc1's role in HT29 cells in regards to NF-κB and MAPK signaling using siRNA is warranted. Discussion Intestinal epithelial cells at mucosal surfaces serve as innate immune sentinels controlling the innate host defense instruction to the immune effector cells inside the body in response to the external environment [1,2]. Previous studies examining pathogenic Salmonella invasion of intestinal epithelial cells demonstrated activation of the proinflammatory gene program and invasion of only a minor portion of the cells [10]. We previously demonstrated that NF-κB is as potently induced in pathogenic Salmonella .sp infected cells similar to those treated with the proinflammatory cytokines that are potent NF-κB activators such as TNFα and IL-1β and that this activity was IKK-mediated [3]. Here we examined how bacterial invasion of only a third of the cells could give rise to NF-κB activity profiles consistent with activation of NF-κB in every cell such as the profile TNFα stimulation provides. We found that bacterial infection activates nuclear translocation of p65 (RelA) in nearly all of the intestinal epithelial cells consistent with the hypothesis that a cell surface receptor was recognizing either a soluble product that bacteria were producing, or a bacterial product on the bacteria, or both. We examined bacterial culture broths and found a bacterial product that was protein in composition and when used to challenge intestinal epithelial cells it potently activated NF-κB DNA binding activity (Fig. 2A). We further purified this protein by gel permeation and anion exchange chromatography and found the protein to be flagellin by electrospray ion trap mass spectroscopy (Fig. 2B &2C and Fig. 3). While our studies were in progress, flagellin was identified as being a potent proinflammatory mediator leading to IL-8 production and secretion [16-18]. We demonstrate in this study that flagellin appears to be exclusively responsible for activating NF-κB in intestinal epithelial cells since flagellin mutant strains do not activate NF-κB (Fig. 4) nor lead to their internalization (Fig. 5B). Furthermore, flagellin challenge of intestinal epithelial cells leads to p65 (RelA) nuclear localization in nearly all of the treated cells (Fig. 5B). Transcription factors like activator protein 1 (AP-1) and NF-κB, which are key regulators/activators of the proinflammatory gene program [57,58] are activated by engagement of the MAPK, SAPK and IKK signaling pathways. We demonstrate that the MAPK, SAPK and IKK signaling pathways activation fails to occur in host cells by infection/exposure to Salmonella strains devoid of flagellin or products in the culture broths derived from those mutant Salmonella strains (Figs. 5A and 6B). We also demonstrate here that combined mutants of both fliC and fljB exhibit a severe lack of invasion (10-4 less than wild-type) and failure to activate stress response signaling, which has not been revealed previously. It is likely that the lack of flagellin production interferes with the functioning of the type III secretion system (TTSS) although flagellin is not known to effect expression of TTSS-required gene products. This hypothesis seems credible since supply of flagellin or bacterial culture components from wild-type Salmonella cultures in trans to the double fliC-/fljB- mutant bacteria fails to compliment their lack of infectivity in gentamycin invsion assays (TT & JD, unpublished observations and see Fig. 5B). In fact, we found the abundance of a subset of Sip and Sop proteins (SipA and SopD) released into the bacterial culture media to be drastically reduced in the flagellin mutant strains used here (TT & JD, unpublished observations). These two proteins have not previously been identified as activators of NF-κB nor are they considered as such here. The TTSS translocates the Salmonella invasion proteins (Sips) and the SopE proteins into the host cell initiating cytoskeletal rearrangements that ultimately lead to bacterial internalization, [11,12,41]. In any event, it is clear that purified flagellin activates a similar cadre of proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated Salmonella. The temporal expression pattern of these genes was found to be remarkably similar (Fig. 6C) indicating that flagellin-mediated temporal activation of the MAPK, SAPK and IKK signaling pathways can suffice for signaling pathways activated by Sips or SopE and SopE2 and largely recapitulates the temporal activation of key proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated Salmonella. The rapid, and potent activation of the MAPK, SAPK and IKK signaling pathways by flagellin was consistent with and indicative of the activation of a cell surface receptor. In this study and in other studies TLR5 has been demonstrated to play an integral role in the recognition of flagellin leading to activation of NF-κB and expression of the IL-8 gene (Fig. 6C) [22,33]. Identification of TLR5 utilized transfection of TLRs 1 – 9 into cell lines which responded poorly to flagellin (this study) or not at all [22,33] and challenging the transfectants with flagellin to identify which TLR responded to this PAMP. Previous studies that identified TLR5 as the receptor for flagellin did not examine the abundance of TLR5 in these cells or account for the lack of TLR5-mediated signaling in response to flagellin [22,33]. We demonstrate here that cells which respond poorly (HeLa and HEK293T) or not at all (T98G) contain TLR5 in at least equivalent abundance as HT29 cells which are highly responsive to flagellin. We propose at least three possibilities to account for this discrepancy, first, this may be due to either lack of TLR5 receptor presence at the plasma membrane and intracellular localization; second, inactivating or detrimental mutations in the TLR5 gene in these cell lines; and lastly, lack of or low abundance of a required co-receptor or adapter protein required for either efficient ligand recognition and/or signaling. These possibilities are currently being investigated. We favor the last possibility since surface biotinylation experiments indicate that TLR5 is present on the cell surface in both flagellin responding cells and in non-responders mentioned above (data not shown). Invocation of the second hypothesis would require inactivating mutations be present in three different cell lines, a highly improbable outcome. How do the findings presented here correlate with events during a "normal" Salmonella infection? We have indicated in this study that defective type III secretion system functioning leads to loss of host cell infectivity and underscores the importance of this system in the normal course of infection. In the in vivo setting, polarized epithelial cells express TLR5 on the basolateral surface [48] and flagellin can only reach the receptor either after either breaching the tight junction barrier by physical damage or by loosening of the junctions in response to Sips and Sops delivered into the intestinal epithelial cells by the TTSS or by delivery of flagellin across the intestinal epithelial cell by the bacteria itself [17,59-61]. This scenario would imply the main function of the type III secretion system would be to trigger stress response signaling facilitating invasion and lead to loosening the tight junctions and result in flagellin/ flagellated bacteria to passing through the junctions and infected cells allowing access the basolateral surface and then systemic dispersion. TLR5 on the basolateral surface of the intestinal epithelial cells, in response to flagellin, could then lead to activation of NF-κB and the proinflammatory gene program and host protection. This model is consistent with activation of the proinflammatory gene program observed in response to flagellated Salmonella sp. infection in many reports too numerous to cite here and would allow the innate host defense system a fail-safe way to recognize pathogen exposure. In instances where infection of intestinal epithelial cells by naturally occurring non-flagellated Salmonella occurs, a strong proinflammatory response would not initially be presented but the Salmonella would instead lead to systemic infection as is the case in chickens with S. galinarum and S. pollorum and result in typhoid-like disease [62]. Infection of chicken epithelial cells does not lead to proinflammatory gene expression by these non-flagellated pathogens but does when infected with S. typhimurium or S. dublin [62]. Argument for the existence of an additional TLR5 co-receptor/adapter being in limited abundance or absent might be in evidence from the transfection results presented in Table 1 which demonstrated that overexpression of cell surface localized FLAG-tagged TLR5 only resulted in slightly over a two-fold increase in NF-κB reporter gene expression in response to flagellin. If only TLR5 was required for activation of the signaling pathway should not a much more robust response been observed? We have also used DN-TLR5 transfections and NF-κB-dependent reporter gene assays or overexpresssion of DN-TLR5 using recombinant adenoviruses and analysis of resulting NF-κB DNA binding activity in response to flagellin to examine its effectiveness to completely inhibit TLR5-mediated flagellin activation of NF-κB. We have found it difficult to gain more than a fifty-percent reduction in either reporter gene activation or NF-κB DNA binding activity in HT29 cells (TT, AD & JD, unpublished observations). These results suggest that the resting TLR5 signaling complex may be quite stable as has recently been suggested [63]. Should the endogenous TLR5 signaling complex be extremely stable it would therefore be expected that titration of a required pre-stimulus bound adapter or co-receptor away would be inefficient and this is what we have observed. Expression of a DN-MAL (TIRAP), a MyD88-related TLR adapter [64,65] had little to no effect on flagellin-mediated NF-κB activity in transient transfection NF-κB reporter gene assays (TT & JD, unpublished observations). Recently, TLR5 has been shown to bind flagellin [66-68] and that this is likely a direct interaction due to failure of the human TLR5 to respond to a purified flagellin derived from a mouse-specific Salmonella strain [68]. These observations still do not preclude the existence of a co-receptor or adapter that is critical for signal transmission. Detailed biochemical characterization of the TLR5 signaling complex will resolve this issue. Muc1, a recently described flagellin interacting membrane protein by virtue of its ability to trigger activation of the MAPK pathway in response to flagellin exposure [55] was considered a viable candidate for such a co-receptor but our observations suggest that it can not serve as the putative TLR5 co-receptor as it is expressed at similar levels in flagellin non-responding cell lines examined here as it is in A549 cells which respond strongly to flagellin and both cell line types express similar levels of TLR5 (Fig. 9). Conclusion In conclusion, our data clearly demonstrates that flagellin can act as the major determinant in activating key stress response signaling pathways and proinflammatory gene program expression in a temporal and qualitative fashion as observed during infection of intestinal epithelial cells by wild-type Salmonella sp. that express flagellin, a point that was not well established until this study. In addition, expression of the fli C gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express fli C are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in trans cannot restore the ability of the fli C mutant bacteria to invade intestinal epithelial cells. Flagellin is "sensed" by TLR5 and in response propagates signaling pathways culminating in potent proinflammatory gene expression. Interestingly we found that TLR5 is expressed in weakly responding and also in some flagellin non-responding cells, 293T, HeLa and T98G cells respectively at levels similar to cells such as HT29 and A549 cells that respond strongly to flagellin and can be found on the cell surface, raising a strong possibility that productive TLR5 signaling may require an additional factor/adaptor other than those already known to be key in the IL-1 signaling pathway, which shares extensive similarities to the TLRs signaling pathways. Methods Materials Human tumor necrosis factor alpha (TNFα) and human IL-1 were purchased from R&D Systems (Minneapolis, MN). Tris [hydroxymethyl]aminomethane (Tris) was purchased from Fisher Scientific (Fairlawn, NJ). Fetal calf serum was purchased from US Biotechnologies Inc. (Parkerford, PA). Para-nitro-phenylphosphate (PNPP) was purchased from Aldrich Chemical (Milwaukee, WI). The Polyacrylamide gel electrophoresis (PAGE) supplies: acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). Dulbecco's modified essential medium (DMEM), DMEM:F12, phosphate buffered saline (PBS), glutamine, penicillin G, streptomycin, amphotericin B, and Grace's Insect medium were purchased from Invitrogen (Carlsbad, CA). Luria Broth (LB) was purchased from Becton Dickson and Co (Sparks, MD). The protease inhibitors: aprotinin, bestatin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cal Biochem (La Jolla, CA). Protease inhibitor cocktail contained 10 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/ml bestatin, and 1.7 μg/ml pepstatin A. Phorbol 12-myristate 13 acetate (PMA), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes), anisomycin, and 2-[N-morpholino]ethanesulfonic acid (MES) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Sigma Chemical or Fisher Scientific unless stated otherwise. Cell culture HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2 atmosphere. T84 colorectal carcinoma cells (ATCC CCL-248) were cultured in DMEM:F12 with 2 mM glutamine, 5% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2 atmosphere. H5 insect cells (Invitrogen) were cultured in Grace's medium with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml amphotericin B at 28°C. MyD88-/- & TLR2-/-/TLR4-/- double knockout cells were obtained from Shizuo Akira and Osamu Takeuchi (Univ. of Osaka, Japan) and grown in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO2 atmosphere. Bacterial strains Salmonella typhimurium strain SJW1103 (FliC, phase 1 flagellin, stabilized) [69] is a wild-type Salmonella typhimurium and can only express the Phase I fliC flagellin, SJW86 (SJW1103 FliC::TN10), and SJW134 (SJW1103 FliC and FljB deletions) were obtained from Robert Macnab (Yale Univ., Conn) and have been described [70]. Salmonella serovar dublin strain 2229, strain SE1 (2229 SopE mutant), strain SB2 (2229 SopB mutant), and SE1SB2 (2229 SopE and SopB mutant) were obtained from Edward Galyov (Compton Laboratory, Berkshire, UK) and have been described [14,15]. Salmonella strains for stimulation were grown in LB at 37°C without agitation for 16 hours, centrifuged at 6,000 × g for 1 minute, gently washed with PBS, and gently suspended in DMEM to maintain cells with attached flagella. Plasmid pFM10.1 (ampicillin resistance), encodes a green fluorescent protein (GFP) expressed after the Salmonella host is internalized by mammalian cells, obtained from Stanley Falkow (Stanford Univ., Stanford, CA) [10,34] and was transformed into strains SJW1103 and SJW134 by electroporation. Strains containing pFM10.1 were designated SJW1103G and SJW134G. Preparation and analysis of Salmonella cell free culture supernatant Native flagellin was harvested from S. dublin 2229 or S. typhimurium SJW1103. Starter cultures were grown in Luria broth (LB) for 18 hours at 37°C with aeration, diluted 1:5000 in fresh LB, and grown for 12 hours under the same conditions. All subsequent procedures were performed at 4°C. Cells were removed from the medium by centrifugation at 10,000 × g for 5 min and discarded. The supernatant containing flagellin was filtered through a 0.8 micron filter (Millipore, Bedford, MA) to remove residual cells. Supernatant was concentrated 100 fold using an Amicon 100 kiloDalton (kDa) cutoff membrane (Millipore). Initial studies used concentrated culture supernatant from S. dublin strain 2229 that was treated with DNase, RNase, Protease K, boiled for 20 min or 100 mM DTT at 37°C for 2 hours and used for stimulation of cultured cells. Concentrated S. typhimurium 1103 bacterial culture supernatant was washed 4 times by 1:10 dilution with 50 mM MES, pH 6.0, 50 mM NaCl and re-concentrated. Material not retained by the 100 kDa membrane was discarded. Washed culture supernatant was fractionated by gel permeation or anion exchange chromatography for analysis. For long-term storage, washed culture supernatant was supplemented with protease cocktail and stored at -20°C. Fractionation by gel permeation chromatography was performed with a Superose 12HR column (Pharmacia) on a Bio-Logic system (Bio-Rad). One-half mililiter of 100× washed supernatant (equivalent of 50 ml original culture supernatant) was separated on the column at 0.4 ml/minute in 50 mM Hepes, pH 7.4, 200 mM NaCl. Fractions (0.5 ml) were collected, and 50 μl was fractionated by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Thirty microliters of each fraction was used for stimulation of HT29 cells (60 mm dishes) for 45 min and NF-κB DNA binding activity in the resulting whole cell extracts extracts were assayed by EMSA. The column was standardized with catalase (232 kDa), aldolase (158 kDa), abumin (67 kDa), ovalbumin (43 kDa), and Chymotripsinogen A (25 kDa), all obtained from Amersham-Pharmacia. Fractionation by anion exchange chromatography was performed with Poros HQ matrix (2 ml column, PerSeptive Biosystems, Farmingham, MA) on a Bio-Logic system. Five mililiters of 100× washed supernatant (equivalent of 500 ml original culture supernatant) was separated at 1 ml/minute in 50 mM Hepes, pH 7.4, and a NaCl gradient from 50–500 mM. Fractions were collected and 5 μl of each fraction was examined by 10% SDS-PAGE. Proteins were fractionated on duplicate 10% SDS-PAGE precast gels (BioRad). One gel was stained with Bio-Safe Coomassie (Bio-Rad) and the protein bands were isolated for Mass Spectroscopy analysis (CCF Mass spectroscopy core facility) from the other identical non-stained gel, by electro-elution with a whole gel eluter (Bio-Rad) and SDS was removed with SDS-Out (Pierce, Rockland, IL) per the manufacturers directions. Proteins isolated from bands B1 to B6 were acetone precipitated by addition of 20 μg Aprotinin and 5 μg of BSA to each eluted fraction, ice-cold acetone (-20°C) was added to 80%, mixed well and precipitated overnight at -20°C. Proteins were pelleted by centrifugation at 14,000 × g in the cold for 30 min, acetone/liquid was removed and the pellets washed 2× with 1 ml acetone (-20°C). After removal of the acetone, protein pellets were air dried and then resuspended and denatured in 5 μl of 6 M guanidinium hydrochloride (Gu-HCl) at room temperature for 30 min. Resuspended proteins were two-fold serially diluted in DMEM to a final Gu-HCl concentration of 55 mM to renature the proteins. Two hundred fifty microliters of individual renatured proteins/DMEM were added per ml to HT29 cells (60 mm dishes) and whole cell extracts were prepared 45 min after stimulation and were assayed for NF-κB DNA binding activity by EMSA. Purification of flagellin (purified flagellin) The washed and concentrated culture supernatant from S. typhimurium 1103 containing flagellin was boiled for 20 minutes and precipitants removed by centrifugation at 15,000 × g. The supernatant containing flagellin was diluted 1:2 with 50 mM MES, pH 6.0, 50 mM NaCl and mixed with 2 ml Poros SP cation exchange matrix (PerSeptive Biosystems) per 1 liter of original culture. The Poros SP matrix was prepared as a 50% slurry and equilibrated with 50 mM MES, pH 6.0. The flagellin preparation and matrix were mixed on a roller at 12 to 14 RPM for 2 hours. The matrix along with bound contaminants was removed by filtration through a 0.85 micron filter and discarded, flagellin failed to bind to the cation exchange matrix at pH 6.0 and eluted in the flowthrough and was collected. The pH of the flowthrough was adjusted by five-fold dilution of the sample with 50 mM Hepes, pH 7.8, 50 mM NaCl, and loaded onto a Poros HQ anion exchange column (2 ml column, PerSeptive Biosystems) equilibrated with 50 mM Hepes, pH 7.4, 50 mM NaCl. The column was washed with 2 volumes 50 mM Hepes, pH, 7.4, 50 mM NaCl, and eluted with a 10 column volume linear gradient of 50–500 mM NaCl in 50 mM Hepes, pH 7.4. Flagellin eluted from the column between 200–275 mM NaCl. Fractions containing flagellin were pooled and concentrated. The preparation was determined to be pure by electrophoresis of 5 μg protein by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Samples were stored at -80°C in 50 mM Hepes, pH 7.4, approx 225 mM NaCl, 10% glycerol and protease cocktail. A 4 liter preparation of culture supernatant yielded 2 mg purified flagellin. In-gel tryptic digestion and protein identification by LC-MS Gels were fixed and stained (Bio-Safe Blue, BioRad). All of the following procedures were performed by the CCF Mass spectroscopy core facility. Excised gel bands were reduced (100 mM DTT), and alkylated (100 mM iodoacetamide). Proteins in the gel bands were digested with modified trypsin (Promega, 20 μg/mL) with an overnight incubation at 37°C. Tryptic peptides were extracted from the gel with 50% acetonitrile, 0.1% acetic acid, concentrated in a SpeedVac (Thermo Savant) to remove acetonitrile, and reconstituted to 20 uL with 0.1% acetic acid. Extracted peptides were subjected to reversed phase (50 uM ID packed with Phenomenex Jupiter C18, 6 cm capillary column) liquid chromatography (2%–70% solvent B; Solvent A, 50 mM acetic acid, aqueous, Solvent B acetonitrile), coupled to a Finnigan LCQ DECA ion trap mass spectrometer for peptide sequencing, as described [38]. Preparation of GST-IκBa1-54 and GST-cJUN1-79 kinase substrates IκBα amino acids 1 to 54 fused to GST or cJUN amino acids 1–79 fused to GST were prepared as previously described [37-39] and stored in kinase buffer (20 mM Hepes, pH 7.6, 10 MM MgCl2, 10 mM NaCl, 2 mM beta-glycerophosphate, 10 mM PNPP). Preparation of cells for microscopy HT29 cells for microscopic examination were grown in 6 well plates on sterile cover slips to a density of 50–75%. Cells were stimulated as described above. After stimulation, cover slips with HT29 cells were washed 2 times with ice cold PBS and fixed with 4% w/v formalin at room temperature for 20 minutes. Cells were washed 4 times with PBS prior to mounting for visualization of Salmonella invasion. Cover slips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and cover slips sealed to slides. Cells for antibody staining were treated with absolute methanol for 20 minutes following formalin fixation, then washed 3 times with PBS supplemented with 0.1% BSA (PBSB) and used directly or stored in the cold after azide was added to 0.02%. For p65(RelA) localization, cells on coverslips were blocked for 1 h at 37°C with PBS supplemented with 1% BSA. The PBSB was removed, washed once with PBSB and coverslips were placed cell-side down onto 150 μl of p65 antibody (Zymed, South San Francisco, CA) diluted 1:1500 in PBSB on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 3 × 5 min with PBSB. Coverslips were then removed and placed cell-side down onto 150 μl of FITC-labeled donkey anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) (1:300 in PBSB) on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 5 × 5 min with PBSB, removed and placed cell-side down onto slides mounted with Vectashield (Vector Laboratories, Burlingame, CA) with DAPI and then sealed. NF-κB localization was determined by indirect immunofluorescence. Samples were observed on a Leica DMR upright microscope (Leica Microsystems Inc., Heidelberg, Germany) at 400× with oil immersion and equipped with FITC and UV filters. Images were collected with a MicroMax RS camera (Princeton Instruments Inc., Princeton, NJ), and Image Pro plus, version 4.5, software (Media Cybermetics Inc., Carlsbad, CA). Color enhancements were performed with Image Pro plus software. Visible light plus color overlays for Fig. 1 and Fig. 5B were performed with MetaMorph Software (Universal Imaging Corp., Downington, PA). Bacterial infection and cell stimulation Mouse embryo fibroblasts (MEFs) or HT29 cells were grown in DMEM as above to a density of 90% prior to stimulation. All cells were washed with warm PBS and supplemented with DMEM without serum or antibiotics in preparation for stimulation. Cells were stimulated with; 10 ng/ml TNFα, 1 μg/ml flagellin unless specified otherwise, 20 μg/ml Anisomycin, 12.5 ng/ml PMA, or 108 Salmonella/ml at 37°C for desired times and extracts prepared as below. Cells harvested beyond one hour were washed with warm PBS and supplemented with warm DMEM, 2 mM glutamine, and 200 ug/ml gentamycin after 1 hour and returned to 37°C until extract preparation desired. Whole cell extract preparation Cells were washed with ice-cold PBS and all subsequent steps carried out at 4°C or on ice. Cells were scraped from the dish in ice-cold PBS, and collected by centrifugation at 1000 × g for 1 minute. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 25 mM beta-glycerol phosphate, 25 mM NaF, 10 mM PNPP, 10 % glycerol, 0.5 mM sodium orthovanadate, 0.5% nonidet-40 (NP-40), 5 mM benzamidine, 2.5 mM metabisulfite, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail as described [3]. Electromobility shift assays (EMSA) NF-κB DNA binding assays were carried out as previously described [3,35,38]. Anti-p65 antibody (Zymed, South San Francisco), anti-p50 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), and anti-STAT3 antibody (Santa Cruz) were used for EMSA supershifts. Invasion assay HT29 cells, 90–95% confluent in 35 mm round dishes, were prepared for stimulation as above and treated with a 1 ml suspension of Salmonella SJW1103 or SJW134 or left untreated in triplicate as above. After one hour, HT29 cells were washed 4× with warm PBS, supplemented with warm DMEM, 2 mM glutamine, and 200 μg/ml gentamycin, and incubated at 37°C for 4 hours. Cells were then harvested as above and lysed by suspension in 1 ml sterile distilled water. Ten-fold serial dilutions were prepared in PBS and 100 μl of each dilution was plated on LB agar plates and grown at 37°C for 20 hours. Colonies were counted and averaged. Kinase assays Whole cell extracts (250 μg) were supplemented with 150 μl of Buffer A (20 mM Hepes, pH 7.9, 20 mM beta-glycerophosphate, 10 mM NaF, 0.1 mM orthovanadate, 5 mM PNPP, 10 mM 2-mercaptoethanol, 0.5 mM PMSF, and protease inhibitor cocktail), and immuno precipitation kinase assays carried out as described [3] using either IKKα monoclonal antibody (PharMingen – Becton Dickson), anti-JNK1 (Santa Cruz Biotechnologies, Santa Cruz, CA), or anti-hemagglutinin (HA) epitope antibody (Covence Antibodies, Princeton, NJ) as indicated. Protein G immunopellets were collected by centrifugation at 500 × g for 30 sec, washed 3 times with Buffer B (Buffer A plus 250 mM NaCl), and one time with Buffer C (Buffer A plus 50 mM NaCl and 10 mM MgCl2). Immunopellets were resuspended in 30 μl Kinase buffer with 0.1 mM orthovanadate, 50 μM "cold" ATP, 5 μCi γ-32P-ATP, 2 mM DTT, and 2 μg of soluble GST-IκBα1–54 or GST-cJUN1-79, and incubated at 30°C for 30 minutes. Reactions were stopped by the addition of 15 μl 4× SDS-PAGE loading buffer, heated at 95°C for 5 minutes, and resolved on 10% SDS-PAGE gels by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (Bio-Rad) to visualize protein bands, rinsed, photographed then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) to detect substrate phosphorylation. Immunoblotting Protein samples (40 μg) were resolved by SDS-PAGE on a 10% acrylamide gels by standard procedures, and proteins transferred to PVDF membrane (Millipore) and probed with antibodies as described [3]. Membranes were washed 3× briefly with TBST, incubated with a 1:1000 dilution (1:800 for anti-TLR5) of the primary antibody in TBST, 1% non-fat milk for 1 hour, washed 3 × 5 min with TBST, and then incubated with a 1:2000 dilution of the appropriate HRP-conjugated secondary antibody in TBST, 0.5% non-fat milk for 1 hour. Primary antibodies used were: anti-IKKα/β (H-470, Santa Cruz), anti-JNK1, anti-ERK2 (K-23, Santa Cruz), anti-phospho-ERK (E-4, Santa Cruz), anti-p38MAPK (Cell Signaling Technologies, Beverly, MA), anti-phosopho-p38MAPK (Cell Signaling), anti-TLR5 (H-127, Santa Cruz), anti-muc1 (H-295, Santa Cruz) and anti-actin (C-11, Santa Cruz). Secondary antibodies used were: anti-mouse IgG HRP conjugate (Amersham-Pharmacia), anti-rabbit IgG HRP conjugate (Amersham-Pharmacia), anti-goat IgG-HRP conjugate (Santa Cruz). HRP activity was detected by ECL (Amersham-Pharmacia) as per manufacturers instructions, on Kodak X-OMAT AR film. Construction of dominant-negative TLRs All DN-TLRs were constructed using PCR. The universal 5' primer consisted of a 5'KPN I restriction site followed by sequences encoding the kozak sequence, translational start site, and preprotrypsin leader sequence of pCMV-1 (Sigma) that all the wild-type TLRs were initially cloned into. The 3' anti-sense (AS) primers were human TLRgene-specific primers (sequences available upon request) that created a stop codon immediately after a conserved tryptophan in Box 9 of the TLR TIR homology domain according to Bazan [71], thus creating carboxy terminus deletions. The 5' end of the AS primer contained a number of convenient restriction sites to allow directional cloning. PCR was performed with turbo-Pfu polymerase (Stratagene, La Jolla, CA) using standard procedures on individual wild-type TLR pCMV-1 plasmid DNAs (5 ng each, kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) [48,49] with the 150 ng each of the universal 5' sense primer and individual gene-specific TLR 3' primers. PCR products were cleaned-up with PCR cleanup kit (Qiagen, Germany) digested with appropriate restriction enzymes, gel purified and then ligated into the mammalian expression vector pCDNA3.1 (Invitrogen). Positive clones were sequenced to verify the mutations and tested for expression in transient expression assays and detected on immunoblots by probing with anti-FLAG M2 monoclonal antibody (Sigma). All wild-type and DN-TLR alleles are amino terminus FLAG epitope-tagged. Transfections HT29 cells were transfected with Lipofectamine Plus (Invitrogen) as previously described [3]. In transfections monitoring reporter gene expression, transfections were performed at least three times in 6 well dishes in triplicate with the total DNA mass kept constant at 4 μg (2 μg effector plasmid DNA, 100 ng 2× NF-κB Luc reporter gene, 50 ng pRL-TK, a thymidine kinase promoter driven Renilla luciferase normalization reporter and 1.85 μg pCDNA3.1 plasmid DNA as bulk filler DNA) and fire-fly luciferase expression was normalized to Renilla luciferase expression using the dual-luciferase assay (Promega, Madison, WI). Fold inductions were calculated and values between experiments did not vary more than 15%, a representative experiment is presented. Transfection of 293T cells was performed with lipofectamine 2000 (Invitrogen) in 6-well dishes in triplicate as per the manufacture's protocol. TLR expression plasmids were added at 2 μg/well, and NF-κB and normalization control plasmids were as above with HT29 cells and pCDNA3.1 plasmid DNA as bulk filler DNA to a final DNA mass of 4 μg/well. Fold inductions were calculated and values between experiments (N of 3) did not vary more than 10%, a representative experiment is presented. Real Reverse Transcription and Real Time PCR (RT2PCR) Cells (N = 3) were stimulated 3 hours at 37°C with TNFα or FliC or left untreated and harvested for total RNA isolation. Total cellular RNA was extracted from cells with Trizol reagent (Invitrogen) [3] and reverse transcribed with ReactionReady first strand cDNA synthesis kit (SuperArray Bioscience Corp., Fredrick, MD). RNA (2.5 ug per 20 ul reaction) was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase per manufacturer specified conditions. Controls without reverse transcriptase (minus RT) was also generated for each RNA sample. RT2PCR was performed with an iCycler (Bio-Rad) to quantify TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT2PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) per manufacturers instructions in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 15 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT2PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Real-time PCR analysis was performed using SYBR-green (Perkin-Elmer) according to manufacture's instructions with the specific primer pairs indicated above and primer pairs for 18S ribosomal RNA as reference RNA (Classic 18S primer pairs – Ambion Inc). Cycle time (Ct) was measured using the iCycler™ and its associated software (Bio-Rad). Relative transcript quantities were calculated by the ΔΔCt method using 18S ribosomal RNA as a reference amplified from samples using the Classic 18S primer pairs from Ambion, Inc (Austin, TX). Normalized samples were then expressed relative to the average ΔCt value for untreated controls to obtain relative fold-change in expression levels. Fold change in mRNA expression was expressed as 2ΔΔCt. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. Values for fold-induction varied less than 5% among replicates. Abbreviations The abbreviations used are: FBS, fetal bovine serum; IL-1, interlukin-1, SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EMSA, electromobility shift assay; IB, immunoblot; KA, kinase assay; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TNFα, tumor necrosis factor α; NF-κB, nuclear factor kappa B; IKK, Ikappa B kinase; IκB, Ikappa B; PCR, polymerase chain assay; RT-PCR, reverse transcription polymerase chain assay; Gu-HCl, guanidinium hydrochloride ; MAPK, mitogen activated protein kinase; SAPK, stress-activated protein kinase; ERK, extracellular regulated kinase; TLR, toll-like receptor; DN, dominant-negative; JNK, Jun N-terminal kinase; AP-1, activator protein-1; MEF, mouse embryo fibroblast; WCE, whole cell extract; IEC, intestinal epithelial cell; MCP1, macrophage chemoattractant protein 1; TTSS, type III secretion system; Sip, Salmonella invasion protein; PMA, phorbol 12-myristate 13 acetate; PNPP, para nitrophenyl phosphate; TK, thymidine kinase; BF, bright field; NP-40, nonidet-40; NRS, normal rabbit serum; IN, input; Ct, cycle time. Authors' contributions TT and AD initiated the study and performed the majority of the experiments and contributed equally and were assisted by NK and JL. MD constructed a number of DN-TLRs and JD developed the study, provided funding support, oversaw the project and also constructed a number of mutant TLRs. Acknowledgements We would like to thank Drs.E.Galyor (Compton Laboratory, Berkshire (UK)), Drs. R.M. Macnab (Yale University, New Haven, Conn), S. Mizel (Wake Forest University, Winston-Salem, NC), and M. Kagnoff (UCSD, La Jolla, CA) for bacterial strains. We would also like to thank S. Akira and O. Takeuchi (Osaka University, Osaka, JA) for the gift of MyD88 and TLR 2/4 double knockout cell lines. We also would like to thank S. Falkow (Stanford University, Stanford, CA), M. Karin (UCSD, La Jolla, CA), R. Medzhitov (Yale University, New Haven, Conn) and R. Ulevitch (TSRI, La Jolla, CA) and V. Dixit (then at Univ. of Michigan, Ann Arbor, MI) for gifts of plasmids. We would also like to thank B. Williams (CCF, Cleveland, OH) for thoughtful discussions and support on this project. This work was supported in part by grants from the National Institutes of Health, CA84406 (to J.A.D.) and the U.S. Army, DAMD 17-01-C-0065 (to B. Williams). ==== Refs Kagnoff MF Eckmann L Epithelial cells as sensors for microbial infection J Clin Invest 1997 100 6 10 9202050 Eckmann L Kagnoff MF Fierer J Intestinal epithelial cells as watchdogs for the natural immune system Trends Microbiol 1995 3 118 120 7773590 10.1016/S0966-842X(00)88894-0 Elewaut D DiDonato JA Kim JM Truong F Eckmann L Kagnoff MF NF-kappa B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria J Immunol 1999 163 1457 1466 10415047 Witthoft T Eckmann L Kim JM Kagnoff MF Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells Am J Physiol 1998 275 G564 G571 9724270 Eckmann L Stenson WF Savidge TC Lowe DC Barrett KE Fierer J Smith JR Kagnoff MF Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin h synthase-2 expression and prostaglandin E2 and F2 alpha production J Clin Invest 1997 100 296 309 9218506 Jung HC Eckmann L Yang SK Panja A Fierer J Morzycka-Wroblewska E Kagnoff MF A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion J Clin Invest 1995 95 55 65 7814646 Hardt WD Chen LM Schuebel KE Bustelo XR Galan JE S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells Cell 1998 93 815 826 9630225 10.1016/S0092-8674(00)81442-7 Hobbie S Chen LM Davis RJ Galan JE Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells J Immunol 1997 159 5550 5559 9548496 Chen LM Hobbie S Galan JE Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear responses Science 1996 274 2115 2118 8953049 10.1126/science.274.5295.2115 Valdivia RH Hromockyj AE Monack D Ramakrishnan L Falkow S Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions Gene 1996 173 47 52 8707055 10.1016/0378-1119(95)00706-7 Galan JE Salmonella interactions with host cells: type III secretion at work Annu Rev Cell Dev Biol 2001 17 53 86 11687484 10.1146/annurev.cellbio.17.1.53 Galan JE Collmer A Type III secretion machines: bacterial devices for protein delivery into host cells Science 1999 284 1322 1328 10334981 10.1126/science.284.5418.1322 Kubori T Galan JE Salmonella type III secretion-associated protein InvE controls translocation of effector proteins into host cells J Bacteriol 2002 184 4699 4708 12169593 10.1128/JB.184.17.4699-4708.2002 Bakshi CS Singh VP Wood MW Jones PW Wallis TS Galyov EE Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells J Bacteriol 2000 182 2341 2344 10735884 10.1128/JB.182.8.2341-2344.2000 Wood MW Rosqvist R Mullan PB Edwards MH Galyov EE SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry Mol Microbiol 1996 22 327 338 8930917 10.1046/j.1365-2958.1996.00116.x Eaves-Pyles T Szabo C Salzman AL Bacterial invasion is not required for activation of NF-kappaB in enterocytes Infect Immun 1999 67 800 804 9916093 Gewirtz AT Simon PO JrSchmitt CK Taylor LJ Hagedorn CH O'Brien AD Neish AS Madara JL Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response J Clin Invest 2001 107 99 109 11134185 Gewirtz AT Rao AS Simon PO JrMerlin D Carnes D Madara JL Neish AS Salmonella typhimurium induces epithelial IL-8 expression via Ca(2+)-mediated activation of the NF-kappaB pathway J Clin Invest 2000 105 79 92 10619864 McDermott PF Ciacci-Woolwine F Snipes JA Mizel SB High-affinity interaction between gram-negative flagellin and a cell surface polypeptide results in human monocyte activation Infect Immun 2000 68 5525 5529 10992449 10.1128/IAI.68.10.5525-5529.2000 Ciacci-Woolwine F Blomfield IC Richardson SH Mizel SB Salmonella flagellin induces tumor necrosis factor alpha in a human promonocytic cell line Infect Immun 1998 66 1127 1134 9488405 Moors MA Li L Mizel SB Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin Infect Immun 2001 69 4424 4429 11401982 10.1128/IAI.69.7.4424-4429.2001 Gewirtz AT Navas TA Lyons S Godowski PJ Madara JL Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression J Immunol 2001 167 1882 1885 11489966 Akira S Toll-like receptors and innate immunity Adv Immunol 2001 78 1 56 11432202 10.1016/S0065-2776(01)78001-7 Akira S Hemmi H Recognition of pathogen-associated molecular patterns by TLR family Immunol Lett 2003 85 85 95 12527213 10.1016/S0165-2478(02)00228-6 Medzhitov R Janeway C Jr The Toll receptor family and microbial recognition Trends Microbiol 2000 8 452 456 11044679 10.1016/S0966-842X(00)01845-X O'Neill L Specificity in the innate response: pathogen recognition by Toll-like receptor combinations Trends Immunol 2001 22 70 11286699 10.1016/S1471-4906(00)01842-1 Hajjar AM O'Mahony DS Ozinsky A Underhill DM Aderem A Klebanoff SJ Wilson CB Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin J Immunol 2001 166 15 19 11123271 Ozinsky A Underhill DM Fontenot JD Hajjar AM Smith KD Wilson CB Schroeder L Aderem A The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors Proc Natl Acad Sci U S A 2000 97 13766 13771 11095740 10.1073/pnas.250476497 Kawasaki K Akashi S Shimazu R Yoshida T Miyake K Nishijima M Involvement of TLR4/MD-2 complex in species-specific lipopolysaccharide-mimetic signal transduction by Taxol J Endotoxin Res 2001 7 232 236 11581576 10.1179/096805101101532738 Nagai Y Akashi S Nagafuku M Ogata M Iwakura Y Akira S Kitamura T Kosugi A Kimoto M Miyake K Essential role of MD-2 in LPS responsiveness and TLR4 distribution Nat Immunol 2002 3 667 672 12055629 Backhed F Normark S Richter-Dahlfors A TLR4-dependent lipopolysaccharide signalling in epithelial cells is independent of extracellular protease activity Cell Microbiol 2002 4 297 303 12027957 10.1046/j.1462-5822.2002.00190.x da Silva CJ Soldau K Christen U Tobias PS Ulevitch RJ Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2 J Biol Chem 2001 276 21129 21135 11274165 10.1074/jbc.M009164200 Hayashi F Smith KD Ozinsky A Hawn TR Yi EC Goodlett DR Eng JK Akira S underhill DM Aerem A The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5 Nature 2001 410 1099 1103 11323673 10.1038/35074106 Valdivia RH Falkow S Fluorescence-based isolation of bacterial genes expressed within host cells Science 1997 277 2007 2011 9302299 10.1126/science.277.5334.2007 DiDonato JA Mercurio F Karin M Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B Mol Cell Biol 1995 15 1302 1311 7862124 Norris FA Wilson MP Wallis TS Galyov EE Majerus PW SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase Proc Natl Acad Sci U S A 1998 95 14057 14059 9826652 10.1073/pnas.95.24.14057 DiDonato JA Assaying for I kappa B kinase activity Methods Enzymol 2000 322 393 400 10914035 10.1016/S0076-6879(00)22038-7 DiDonato JA Hayakawa M Rothwarf DM Zandi E Karin M A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB Nature 1997 388 548 554 9252186 10.1038/41493 Hibi M Lin A Smeal T Minden A Karin M Identification of an oncoprotein and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain Genes Dev 1993 7 2135 2148 8224842 Galkin VE Orlova A VanLoock MS Zhou D Galan JE Egelman EH The bacterial protein SipA polymerizes G-actin and mimics muscle nebulin Nat Struct Biol 2002 9 518 521 12055622 Wallis TS Wood M Watson P Paulin S Jones M Galyov E Sips, Sops, and SPIs but not stn influence Salmonella enteropathogenesis Adv Exp Med Biol 1999 473 275 280 10659368 Muzio M Ni J Feng P Dixit VM IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling Science 1997 278 1612 1615 9374458 10.1126/science.278.5343.1612 Takeuchi O Akira S MyD88 as a bottle neck in Toll/IL-1 signaling Curr Top Microbiol Immunol 2002 270 155 167 12467250 Medzhitov R Preston-Hurlburt P Kopp E Stadlen A Chen C Ghosh S Janeway CA Jr MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways Mol Cell 1998 2 253 258 9734363 10.1016/S1097-2765(00)80136-7 Yoshimura A Lien E Ingalls RR Tuomanen E Dziarski R Golenbock D Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2 J Immunol 1999 163 1 5 10384090 Lien E Sellati TJ Yoshimura A Flo TH Rawadi G Finberg RW Carroll JD Espevik T Ingalls RR Radolf JD Golenbock DT Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products J Biol Chem 1999 274 33419 33425 10559223 10.1074/jbc.274.47.33419 Lien E Means TK Heine H Yoshimura A Kusumoto S Fukase K Fenton Oikawa M Qureshi N Monks B Finberg RW Ingalls RR Golenbock DT Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide J Clin Invest 2000 105 497 504 10683379 Alexopoulou L Holt AC Medzhitov R Flavell RA Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3 Nature 2001 413 732 738 11607032 10.1038/35099560 Chuang TH Ulevitch RJ Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9 Eur Cytokine Netw 2000 11 372 378 11022120 Devary Y Rosette C DiDonato JA Karin M NF-kappa B activation by ultraviolet light not dependent on a nuclear signal Science 1993 261 1442 1445 8367725 Heguy A Baldari CT Macchia G Telford JL Melli M Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction J Biol Chem 1992 267 2605 2609 1531143 Croston GE Cao Z Goeddel DV NF-kappa B activation by interleukin-1 (IL-1) requires an IL-1 receptor-associated protein kinase activity J Biol Chem 1995 270 16514 16517 7622454 10.1074/jbc.270.28.16514 Akashi S Nagai Y Ogata H Oikawa M Fukase K Kusumoto S Kawasaki K Nishijima M Hayashi S Kimoto M Miyake K Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition Int Immunol 2001 13 1595 1599 11717200 10.1093/intimm/13.12.1595 Schromm AB Lien E Henneke P Chow JC Yoshimura A Heine H Latz E Monks BG Schwartz JA Miyake K Golenbock DT Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling J Exp Med 2001 194 79 88 11435474 10.1084/jem.194.1.79 Lillehoj EP Kim H Chun EY Kim KC Pseudomonas aeruginosa Stimulates Phosphorylation of the Epithelial Membrane Glycoprotein Muc1 and Activates MAP Kinase Am J Physiol Lung Cell Mol Physiol 2004 Lillehoj EP Kim BT Kim KC Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin Am J Physiol Lung Cell Mol Physiol 2002 282 L751 L756 11880301 Karin M Takahashi T Kapahi P Delhase M Chen Y Makris C Rothwarf D Baud V Natoli G Guido F Li N Oxidative stress and gene expression: the AP-1 and NF-kappaB connections Biofactors 2001 15 87 89 12016332 Jobin C Sartor RB The I kappa B/NF-kappa B system: a key determinant of mucosalinflammation and protection Am J Physiol Cell Physiol 2000 278 C451 C462 10712233 Wood MW Jones MA Watson PR Siber AM McCormick BA Hedges S Rosquist R Wallis TE Galyov EE The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis Cell Microbiol 2000 2 293 303 11207586 10.1046/j.1462-5822.2000.00054.x McCormick BA The use of transepithelial models to examine host-pathogen interactions Curr Opin Microbiol 2003 6 77 81 12615224 10.1016/S1369-5274(02)00003-6 Sakaguchi T Kohler H Gu X McCormick BA Reinecker HC Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells Cell Microbiol 2002 4 367 381 12067320 10.1046/j.1462-5822.2002.00197.x Kaiser P Rothwell L Galyov EE Barrow PA Burnside J Wigley P Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum Microbiology 2000 146 3217 3226 11101679 Mizel SB Snipes JA Gram-negative flagellin-induced self-tolerance is associated with a block in interleukin-1 receptor-associated kinase release from toll-like receptor 5 J Biol Chem 2002 277 22414 22420 11953430 10.1074/jbc.M201762200 Fitzgerald KA Palsson-McDermott EM Bowie AG Jefferies CA Mansell AS Brady G Brint E Dunne A Gray P harte MT McMurray D Smith DE Sims JE Bird TA O'Neill LA Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction Nature 2001 413 78 83 11544529 10.1038/35092578 Horng T Barton GM Medzhitov R TIRAP: an adapter molecule in the Toll signaling pathway Nat Immunol 2001 2 835 841 11526399 10.1038/ni0901-835 Mizel SB West AP Hantgan RR Identification of a sequence in human toll-like receptor 5 required for the binding of Gram-negative flagellin J Biol Chem 2003 278 23624 23629 12711596 10.1074/jbc.M303481200 Murthy KG Deb A Goonesekera S Szabo C Salzman AL Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro J Biol Chem 2003 Smith KD Andersen-Nissen E Hayashi F Strobe K Bergman MA Barrett SL Cookson BT Aderem A Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility Nat Immunol 2003 4 1247 1253 14625549 10.1038/ni1011 Yamaguchi S Fujita H Sugata K Taira T Iino T Genetic analysis of H2, the structural gene for phase-2 flagellin in Salmonella J Gen Microbiol 1984 130 255 265 6374019 Williams AW Yamaguchi S Togashi F Aizawa SI Kawagishi I Macnab RM Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium J Bacteriol 1996 178 2960 2970 8631688 Rock FL Hardiman G Timans JC Kastelein RA Bazan JF A family of human receptors structurally related to Drosophila Toll Proc Natl Acad Sci U S A 1998 95 588 593 9435236 10.1073/pnas.95.2.588

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==== Front BMC Cardiovasc DisordBMC Cardiovascular Disorders1471-2261BioMed Central London 1471-2261-4-151531765410.1186/1471-2261-4-15Research ArticleDisparities in lipid management for African Americans and Caucasians with coronary artery disease: A national cross-sectional study Massing Mark W [email protected] Kathleen A [email protected] Lori [email protected] Carla A [email protected] Charles M [email protected] Ross J [email protected] Health Care Assessment, Medical Review of North Carolina, Cary, North Carolina, USA2 Departmment of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, North Carolina, USA3 Outcomes Research and Management, Merck & Company, West Point, Pennsylvania, USA4 Institute for Health, Social, and Community Research, Shaw University, Raleigh, North Carolina, USA5 Department of Cardiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA2004 18 8 2004 4 15 15 16 2 2004 18 8 2004 Copyright © 2004 Massing et al; licensee BioMed Central Ltd.2004Massing et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Individuals with coronary artery disease are at high risk for adverse health outcomes. This risk can be diminished by aggressive lipid management, but adherence to lipid management guidelines is far from ideal and substantial racial disparities in care have been reported. Lipid treatment and goal attainment information is not readily available for large patient populations seen in the fee-for-service setting. As a result, national programs to improve lipid management in this setting may focus on lipid testing as an indicator of lipid management. We describe the detection, treatment, and control of dyslipdemia for African Americans and Caucasians with coronary artery disease to evaluate whether public health programs focusing on lipid testing can eliminate racial disparities in lipid management. Methods Physicians and medical practices with high numbers of prescriptions for coronary artery disease medications were invited to participate in the Quality Assurance Program. Medical records were reviewed from a random sample of patients with coronary artery disease seen from 1995 through 1998. Data related to the detection, treatment, and control of dyslipidemia were abstracted from the medical record and evaluated in cross-sectional stratified and logistic regression analyses using generalized estimation equations. Results Data from the medical records of 1,046 African Americans and 22,077 Caucasians seen in outpatient medical practices in 23 states were analyzed. African-American patients were younger, more likely to be women and to have diabetes, heart failure, and hypertension. The low density lipoprotein cholesterol (LDL-C) testing rate for Caucasian men was over 1.4 times higher than that for African-American women and about 1.3 times higher than that for African-American men. Almost 60% of tested Caucasian men and less than half of tested African Americans were prescribed lipid-lowering drugs. Tested and treated Caucasian men had the highest LDL-C goal attainment (35%) and African-American men the lowest (21%). Conclusions Although increased lipid testing is clearly needed for African Americans, improvements in treatment and control are also necessary to eliminate racial disparities in lipid management. Disparities in treatment and goal attainment must be better understood and reflected in policy to improve the health of underserved populations. ==== Body Background Individuals with coronary disease (CAD) are at high risk for subsequent cardiovascular disease events and mortality[1]. Clinical trials have shown that this risk can be substantially reduced though the detection, treatment, and control of dyslipidemia[2,3]. To that end, clinical guidelines have been established for the lipid management of CAD patients[4,5]. Adherence to these guidelines is far from ideal[6,7]. Substantial racial disparities in the diagnosis and management of dyslipidemia have been reported in the general population and among CAD patients [8-16]. Improved lipid management through the diagnosis of dyslipidemia has been a focus of quality improvement programs in the outpatient fee-for-service setting such as Medicare's Health Care Quality Improvement Program[17]. Lipid testing is used as an indicator of lipid management in the fee-for-service setting because testing is a service identifiable in insurance claims data. In contrast, assessment of treatment and goal attainment in the fee-for-service setting requires resource-intensive medical record review which is generally not performed for large national patient populations. This study describes outpatient lipid management for African Americans and Caucasians with CAD seen in medical practices throughout the United States. Data from medical records were examined for indicators of lipid management including lipid testing, lipid-lowering drug prescription, and goal attainment. Our objectives were to characterize lipid management across race-sex groups and evaluate the extent of disparities for the three components of lipid management: detection, treatment, and control. We then discuss the implications of our findings with respect to possible underlying causes and health policies for closing the gap in race-sex lipid management disparities. Methods The Quality Assurance Program The Quality Assurance Program (QAP) is a national program sponsored by Merck & Company conducted during the late 1990's to identify physician practice patterns and to promote evidence-based best practices for the medical management of patients with cardiovascular disease seen in the outpatient setting [18]. The QAP database provides abstracted medical record data collected in two distinct time periods and study populations nationwide. Our analyses were limited to the study population identified in the most recent period of data collection (QAP-II). Patients from QAP-II included in these analyses were seen at participating medical practices from January, 1995 through March, 1998. Medical records were reviewed by Access Medical Ltd (Arlington, VA) using a standardized electronic abstraction tool developed specifically for QAP-II. The QAP database contains data obtained from the medical record including race, sex, date of birth, medical history, and medical procedures. The most recent serum lipid testing results and the most recently recorded prescriptions for lipid-lowering drugs were also determined from the medical record. The medical record of each patient was reviewed only once. Patients were not followed over time. Patient and physician identifying information were not included in the QAP-II database to ensure confidentiality. QAP participant selection Physicians and medical practices throughout the United States with high numbers of prescriptions for medications used in the treatment of cardiovascular disease were invited to participate in the QAP. The specialties of participating physicians included cardiology, internal medicine, family medicine, and endocrinology. Patients with cardiovascular disease were randomly selected within each participating medical practice. Inclusion and exclusion criteria Patients included in the QAP study were at least 21 years of age with CAD and/or heart failure and were seen at least twice in two years by the participating physician. Patients were excluded if the medical record indicated a terminal illness, history of a transplant or awaiting transplant, or deceased. Patients without medical record documentation of CAD were excluded from analysis. The presence of CAD was ascertained during analysis from abstracted medical record data based on medical history, International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes (410–414), and cardiac procedures consistent with CAD (i.e., coronary artery bypass graft, angioplasty, and stent). Only patients with medical record documentation of African-American or Caucasian race were included. To reduce the influence of between-state variation in lipid management from states that contribute little information about African American populations, medical practices were excluded if they were located in states with fewer than 10 African-American patients in QAP-II Indicators of lipid management Measures of detection (lipid testing), treatment (lipid-lowering drug prescription), and control (goal attainment) were the indicators of lipid management considered in this study. Low density lipoprotein cholesterol (LDL-C) testing was measured as the percentage of patients with at least one serum LDL-C value documented in the medical record. The use of lipid-lowering drugs (i.e., "treated" patients) was measured as the percentage of patients with medical record documentation of at least one prescription for a statin (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) or non-statin lipid drug (e.g., gemfibrozil). Goal attainment among those with documented LDL-C values was based on recommended guidelines for patients with coronary artery disease (LDL-C < 100 mg/dL)[5]. Analysis We conducted a cross-sectional analysis of abstracted medical record data obtained from QAP-II. Indicators of lipid management (i.e., LDL-C testing, lipid-lowering drug prescription, and LDL-C goal attainment rates) and potential confounding or explanatory variables were examined within strata of race and sex. Co-morbid conditions including diabetes mellitus, myocardial infarction, heart failure, and hypertension were identified from medical history and diagnosis codes. Logistic regression analyses were performed to evaluate the associations of race and sex with each of three dichotomous lipid management indicators while controlling for multiple confounding and explanatory variables. We accounted for correlations within medical practices using the generalized estimation equation (GEE) method[19]. This method was implemented in generalized linear models with PROC GENMOD of SAS Version 9 (SAS, Inc, Cary, North Carolina) for logistic regression with correlated data[20]. Separate models were run for each indicator of lipid management as the dependent variable. The entire study population was included for logistic regression analyses of LDL-C testing as the dependent variable. Regression analyses with lipid-lowering drug prescription as the dependent variable included only patients with LDL-C tests. Regression analyses for LDL-C goal attainment were limited to patients who received at least one LDL-C test and had a documented prescription for a lipid-lowering drug. The independent variables included in the regression models in addition to race and sex were age, medical history (diabetes mellitus, myocardial infarction, heart failure, hypertension), and geographic region of medical practice. Logistic models predicting lipid-lowering drug prescriptions included a term for serum LDL-C concentration in addition to the above variables. LDL-C concentration was included to control for the severity of hyperlipidemia as a factor in the physician's decision to treat with lipid-lowering drugs. Results Population characteristics A total of 23,123 CAD patients with documented race and sex seen by 1,171 physicians at 238 medical practices in 23 states were included in the study. Of these patients, 1,046 were African American and 22,077 were Caucasian. African-American compared to Caucasian patients were more likely to be women (53% versus 36%). The average age for the study population was 69 years and ranged from 22 to 97 years. Within each race group, women were older than men on average and within each sex group Caucasians were older than African Americans (Table 1). The prevalence of co-morbid conditions was high for all race-sex groups. Despite their younger ages, African Americans were more likely than Caucasians to have diabetes, heart failure, and hypertension. Almost half of African-American women and a third of African-American men had diabetes compared to about a quarter of the Caucasian population. About half of African-American men had heart failure and over three quarters of African-American women had hypertension. Consistent with the geographic distribution of race-specific populations, African-American patients were more likely seen in southern medical practices than elsewhere. Table 1 Characteristics of patients by race and sex.* Characteristic African Americans Caucasians Women (n = 558) Men (n = 488) Women (n = 8,038) Men (n = 14,039) Age (mean ± SEM) 66.8 ± 0.5 62.7 ± 0.6 72.1 ± 0.1 67.1 ± 0.1 Medical History   Diabetes Mellitus 43 33 25 21   Myocardial Infarction 38 47 40 48   Heart Failure 42 48 39 34   Hypertension 78 70 63 52 Region   Northeast 27 25 34 31   Midwest 24 30 31 33   South 42 36 22 23   West 8 8 13 13 Serum Lipid Levels  LDL-C (mean ± SEM mg/dl)† 131.3 ± 2.5 133.0 ± 2.5 124.8 ± 0.6 117.2 ± 0.3  LDL-C at goal (%)† 19 18 25 31 *Percent of race- and sex-specific total unless otherwise specified. †For patients with documented tests having valid values Lipid testing Within sex strata, the percent of patients with LDL-C tests was lower for African-Americans compared to Caucasians (Figure 1). Within race strata the percent of patients with LDL-C tests was lower for women than for men. The LDL-C testing rate for Caucasian men was over 1.4 times higher than that for African-American women and about 1.3 times higher than that for African-American men. Figure 1 Lipid Management among CAD patients. Lipid testing, treatment, and goal attainment rates for African-American and Caucasian women (W) and men (M). Lipid treatment Almost 60% of Caucasian men with LDL-C testing were prescribed lipid-lowering drugs (Figure 1). The proportion of tested Caucasian women receiving these drugs (55%) was similar to, but slightly lower than that for Caucasian men. Less than half of tested African-American men (47%) and women (46%) were prescribed lipid-lowering drugs. Goal attainment Including those prescribed and not prescribed lipid-lowering drugs (Table 1), a quarter of Caucasian women and less than 20% of African-American men and women achieved the recommended LDL-C goal. But a higher proportion (31%) of Caucasian men achieved goal. The mean serum LDL-C concentration for Caucasian men (117 mg/dL) was lower than that for Caucasian women (125 mg/dL) and for African-Americans. Mean LDL-C concentrations were similar for African-American men (133 mg/dL) and women (131 mg/dL). Among patients tested and treated with lipid-lowering drugs about two-thirds or more failed to achieve goal (Figure 1). Tested and treated Caucasian men had the best goal attainment rates (35%). Tested and treated Caucasian women had lower levels of goal attainment than Caucasian men. Goal attainment for Caucasian men and for women of either race exceeded that of African-American men. Only about 1 of 5 African-American men prescribed lipid-lowering drugs achieved LDL-C goal. Logistic regression Results from GEE logistic regression analyses controlling for age, co-morbid conditions, and geographic region (Table 2) were consistent with results from the stratified analyses described above. Relative to Caucasians, African-American men and women were under-tested, under-treated, and less likely to be at goal. Relative to men, regardless of race, women were less likely to be tested. But if tested, women were as likely as men of the same race to receive prescriptions for lipid-lowering drugs. African-American women were the least likely to be tested (Odds Ratio, OR = 0.49), and if tested, African-American men were the least likely to be prescribed lipid drugs (OR = 0.59). Among those tested and treated, African-American men were least likely to be at goal (OR = 0.47). That is, among tested patients who were prescribed lipid-lowering drugs, the odds for goal attainment for Caucasian men were more than twice the odds for African-American men. Table 2 Lipid testing, pharmacologic treatment, and goal attainment among African-American men and women and Caucasian women relative to Caucasian men from logistic regression analyses.* African American Caucasian Women Odds Ratio (95% CI)* Men Odds Ratio (95% CI)* Women Odds Ratio (95% CI)* Men Reference Group LDL-C tested (n = 23,104) 0.49 (0.37,0.64) 0.60 (0.47,0.77) 0.80 (0.74,0.86) 1.00 Lipid drug prescribed (n = 14,499)† 0.62 (0.46,0.83) 0.59 (0.45,0.78) 1.04 (0.95,1.13) 1.00 LDL-C goal attainment (n = 8,336) ‡ 0.55 (0.36,0.82) 0.47 (0.30,0.74) 0.76 (0.68,0.85) 1.00 *Odds ratio and 95% confidence intervals (95% CI) from logistic regression models accounting for within-practice correlations with GEE and controlling for race, sex, age, medical history (diabetes mellitus, myocardial infarction, heart failure, hypertension), and geographic region of medical practice. †Regression model includes serum LDL-C concentration. ‡LDL-C goal attainment (<100 mg/dL) among those with documented LDL-C values and treated with lipid-lowering drugs. Discussion Consistent with previous reports, our findings demonstrate that outpatient lipid management for CAD patients in the late 1990's had much room for improvement and that substantial race-sex disparities existed[8,12,16,21-26]. African Americans experienced markedly lower levels of LDL-C testing than Caucasians and, as a result, they may benefit more than Caucasians from interventions to improve testing. Among those tested, African Americans were less likely to be treated and, if treated, they were less likely to be at goal compared to Caucasians. Marked lipid testing disparities by sex suggest a need for more aggressive testing in women. Much of the information needed to assess lipid management can be found only in the medical record. This information is generally not available to national and local public health programs attempting to implement policies promoting quality improvement in the many and diverse medical practices treating large patient populations. The most readily available data for patients seen in the fee-for-service setting is derived from administrative insurance claims for the reimbursement of costs associated with drugs and services. Insurance coverage for lipid-lowering drugs varies across plans. In contrast, lipid testing for CAD patients is a widely covered service that can be identified using electronic billing data without the need to review patient records in medical practices. For this reason, lipid testing is a focus of national efforts in the Medicare population to improve outpatient lipid management in the fee-for-service setting[17]. Our findings demonstrate that substantial disparities in treatment and goal attainment exist among CAD patients with lipid tests. This implies that current public health programs and policies designed to increase lipid testing alone will have limited impact on lipid management disparities. Substantial disparities in lipid treatment and control will likely persist in the absence of disparities in testing. Underlying causes of inadequate lipid management among CAD patients are multiple and likely vary by race and process of care (i.e., detection, treatment, and control). Factors that limit patient-physician encounters and continuity of care may partially account for racial disparities in lipid management. For example, African-American Medicare consumers with diabetes were more likely to receive outpatient care from emergency departments and had fewer physician visits per year than their Caucasian counterparts[12,27]. The QAP data provide insufficient information to evaluate whether health care access and continuity explain lipid management disparities. A report regarding racial disparities in the use of prescription drugs from the Center for Studying Health System Change provides evidence of other factors explaining racial disparities in lipid management[11]. In this report, Medicare consumers 65 years of age and older were surveyed regarding their ability to obtain prescription drugs. African Americans were more than twice as likely as Caucasians to have not filled a prescription because they could not afford it. More than 16% of Medicare insured African Americans reported that they could not afford to fill at least one prescription in 2001. One-fifth of African Americans and 13% of Caucasians with low income could not afford to fill at least one prescription. During the time period of QAP-II, Medicare did not cover the cost of lipid-lowering drugs. Many Medicare consumers have supplemental insurance that assists with drug costs. In the Medicare population, African Americans were less likely than Caucasians to have supplemental insurance and more likely to be of low income[11]. Barriers to lipid management due to affordability may result in racial disparities if affordability differs by race. Patients who can not afford treatment may be less likely to aggressively pursue it with their physicians and may be less likely to comply with physician recommendations for testing and treatment. Secondary prevention of cardiovascular diseases among CAD patients with pharmacologic agents such as statins has been shown to be cost effective[28]. But drug costs that may exceed $2,000 annually can be beyond the reach of low income and underinsured patients[29]. These costs are more likely a barrier for African Americans than for Caucasians and may contribute to lipid management disparities. The fact that African Americans are more likely to be of low income has greater implications than simply the inability to afford medications. Income is one of several indicators of socioeconomic status correlated with factors related to health including education[30]. Low education has been identified as a factor limiting a patient's personal involvement in lipid management[31]. There is evidence that African Americans are less knowledgeable about cholesterol compared to Caucasians[24]. African Americans may be less aware of the need for lipid management and less likely to pursue it with their physician. Achieving LDL-C goal is challenging for all races, but African Americans may require especially aggressive lipid management accompanied by an enhanced understanding of reasons for failure to achieve goal. A recent report concerning patients with CAD and/or diabetes seen at a Veterans Affairs Medical Center found that African Americans were less likely to achieve lipid goal than Caucasians when prescribed identical doses of the same lipid-lowering drug even though African Americans had more clinic visits and lipid tests[10]. The authors speculate that racial disparities in goal attainment may have occurred due to differences in compliance, lifestyle, and baseline LDL-C (higher for African Americans). In a study of LDL-C lowering with pravastatin in an African-American population, only 13% of patients with a LDL-C goal of 100 mg/dL actually achieved it. Incorrect drug regimen, inadequate lipid monitoring, and compliance problems were thought to have contributed to these goal attainment failures[32]. Additional studies are needed to investigate the underlying causes of lower goal attainment for African Americans receiving treatment for dyslipidemia. Physicians have indicated that oversight is a common reason for failure to adhere to lipid testing guidelines[33]. Whether oversight contributes to racial disparities in lipid management is not known, but oversight can be alleviated by system changes in the medical practice[34]. Awareness is growing that implementation of electronic health records is a necessary component of efforts to improve healthcare quality and prevent medical errors[35,36]. The impact of electronic systems on disparities is an interesting area for future research. Lipid management is also influenced by physician attitudes about guidelines and drug effectiveness[37]. A better understanding is needed of physician attitudes and their relations with healthcare disparities. Physician-patient interactions are influenced by race and cultural factors related to race. African-American and Caucasian patients may differ with respect to cultural perceptions of health and disease and the ability of patients to influence or control their health outcomes[38]. African Americans may be less likely than Caucasians to trust their physicians and racial groups may view their relationship with physicians differently[39]. A survey of adults seen in a managed care setting revealed that African Americans viewed their visits with physicians as less participatory than did Caucasians, but they felt more participatory when seeing a physician of their own race[40]. The relatively poorer lipid management for African Americans compared to Caucasians may be partially explained by racial differences in the prevalence of co-morbid conditions. African Americans in the QAP were more likely than Caucasians to suffer from multiple chronic conditions including diabetes and heart failure. It has been shown that patients with diabetes compared to those without diabetes receive poorer lipid management and are less likely to be at goal[41]. An earlier report from the QAP has shown that patients with CAD and heart failure were less likely to receive lipid testing and cholesterol-lowering drugs than those without heart failure[42]. Co-morbid conditions may hinder lipid management in a number ways. These patients may be more likely to have contra-indications to lipid treatment and to present with acute life-threatening conditions that divert attention away from lipid management. In addition, it has been reported that patients with multiple chronic conditions are less likely to afford medications than those with one or fewer chronic conditions[11]. But even after controlling for several highly prevalent co-morbid conditions in logistic regression analyses, we find that significant lipid management disparities persist. There are a number of limitations in the QAP study design and its population that potentially pose a threat to internal and external validity. These limitations suggest that our findings may not reflect the experiences of the general population. The medical practices included in the QAP were restricted to those writing large numbers of prescriptions for cardiovascular disease drugs. It is difficult to evaluate the impact of this selection bias, but we suspect that medical practices included in the QAP represent the larger and more sophisticated providers of care. Thus, lipid management among QAP participants may be better than that found in the general population. Another limitation to our study is the lack of information characterizing medical practices, physicians, and patients with respect to factors related to race, sex, and lipid management. Because of the unavailability of this information, we could not identify underlying factors potentially explaining our findings. Lipid management data for the QAP patients seeing multiple physicians were unavailable from physicians not participating in QAP and this may have influenced results in an unpredictable manner. Considering the relatively small proportion of the study population identified as African American (<5%), it is likely that African Americans in this study do not represent the national population. We have no specific information about QAP medical practices or their patient populations, but we speculate that African Americans in the QAP were likely receiving better care on average than their counterparts in the general population, especially those with little or no access to outpatient care. If this is true, then racial disparities in lipid management in the general population may be even greater that those suggested by our findings. The time frame for this study includes years 1995 through 1998. Despite changes in guidelines and therapies since that time, our findings remain relevant to current practices. They provide historic context and baseline measures of lipid management disparities necessary to evaluate trends. In addition, they direct attention to the persistent need to provide aggressive treatment to high risk populations with CAD, especially African Americans and women who continue to be underserved. Finally, they highlight the growing view that public health strategies in lipid management must shift focus from testing to treatment and goal attainment[43]. Clearly, a multi-pronged approach including all three elements of the process of care (i.e., detection, treatment, and control) is needed to improve lipid management for all race-sex groups, and particularly for African Americans. Although beyond the scope of this report, the application of conceptual models to the process of care may be useful in understanding how racial disparities arise at each step of the process. Furthermore, policies addressing health promotion and non-medical determinants of health such as socioeconomic status, community environment, and lifestyle choices need also be considered in confronting these disparities[44,45]. Successful lipid management likely depends on a variety of processes that determine the provision of medical services including their availability, accessibility, and acceptability. The substantial racial disparities in lipid management among patients in contact with medical providers suggest that the effectiveness of medical services and patient characteristics play a prominent role in lipid management. Policies promoting appropriate delivery of care through system change as well as those ensuring equal access to care are required to eliminate lipid management disparities in the population of high-risk CAD patients[34,46]. Conclusions Our results suggest that policies and programs focusing solely on the elimination of lipid testing disparities can have only limited benefit in reducing the major disparity in lipid management. The elimination of lipid management disparities will require policies that view untested, untreated, and under-treated individuals as separate populations with unique challenges and solutions[43]. Disparities in testing are just one element in explaining overall disparities in lipid management and ultimately, in cardiovascular outcomes. Future research should address patient, physician, and health system factors that lead to lower rates of testing, treatment and goal attainment for African Americans. Disparities in treatment and goal attainment must be better understood and reflected in policy in order to improve the health of underserved populations through optimal lipid management. List of abbreviations CAD Coronary Artery Disease LDL-C Low-density lipoprotein cholesterol QAP Quality Assurance Program GEE Generalized Estimation Equation Competing interests Analyses were funded by an unrestricted grant from Merck & Co., Inc. No other competing interests are declared. Authors' contributions MM conceived of the study, provided analytic and statistical support, and was the leading contributing author. KF participated in the design of the study and provided analytic support and interpretation of findings. LCE provided critical technical review and major contributions to the discussion section. CS provided conceptual guidance, assistance with QAP project database, and interpretation of analytic findings. CA provided critical review, QAP project experience, and contributions to the presentation and interpretation of findings. RS provided critical review, QAP project experience, and contributions to discussion and interpretation of findings. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The contributions of Dr. Lori Carter-Edwards were funded in part by grants from the Agency for Healthcare Research and Quality (R24HS13353) and the National Center on Minority Health and Health Disparities (R24MD000167). ==== Refs Haffner SM Lehto S Ronnemaa T Pyorala K Laakso M Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction N Engl J Med 1998 339 229 234 9673301 10.1056/NEJM199807233390404 Pyorala K Pedersen TR Kjekshus J Faergeman O Olsson AG Thorgeirsson G. Cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease. A subgroup analysis of the Scandinavian Simvastatin Survival Study (4S) Diabetes Care 1997 20 614 620 9096989 Sacks FM Pfeffer MA Moye LA Rouleau JL Rutherford JD Cole TG Brown L Warnica JW Arnold JM Wun CC Davis BR Braunwald E The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. Cholesterol and Recurrent Events Trial investigators N Engl J Med 1996 335 1001 1009 8801446 10.1056/NEJM199610033351401 Expert Panel on Detection Evaluation,and Treatment of High Blood Cholesterol in Adults National Cholesterol Education Program. Second Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II) Circulation 1994 89 1333 1445 8124825 Expert Panel on Detection Evaluation,and Treatment of High Blood Cholesterol in Adults Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III) JAMA 2001 285 2486 2497 11368702 Aronow WS Underutilization of lipid-lowering drugs in older persons with prior myocardial infarction and a serum low-density lipoprotein cholesterol > 125 mg/dl. Am J Cardiol 1998 82 668 9, A6, A8 9732899 10.1016/S0002-9149(98)00401-9 Massing MW Sueta CA Chowdhury M Biggs DP Simpson R.J.,Jr. Lipid management among coronary artery disease patients with diabetes mellitus or advanced age Am J Cardiol 2001 87 646 9, A10 11230856 10.1016/S0002-9149(00)01447-8 Nelson K Norris K Mangione CM Disparities in the diagnosis and pharmacologic treatment of high serum cholesterol by race and ethnicity: data from the Third National Health and Nutrition Examination Survey Arch Intern Med 2002 162 929 935 11966345 10.1001/archinte.162.8.929 Ayanian JZ Landon BE Landrum MB Grana JR McNeil BJ Use of cholesterol-lowering therapy and related beliefs among middle-aged adults after myocardial infarction J Gen Intern Med 2002 17 95 102 11841524 10.1046/j.1525-1497.2002.10438.x Williams ML Morris MT Ahmad U Yousseff M Li W Ertel N Racial differences in compliance with NCEP-II recommendations for secondary prevention at a Veterans Affairs medical center Ethn Dis 2002 12 S1 62 11913623 Reed MC Hargraves JL Cassil A Unequal Access: African-American Medicare Beneficiaries and the Presciption Drug Gap Center for Studying Health System Change 2003 64 Massing MW Henley NS Carter-Edwards L Schenck AP Simpson R.J.,Jr. Lipid testing among patients with diabetes who receive diabetes care from primary care physicians Diabetes Care 2003 26 1369 1373 12716790 Stewart SH Silverstein MD Racial and ethnic disparity in blood pressure and cholesterol measurement J Gen Intern Med 2002 17 405 411 12133153 10.1046/j.1525-1497.2002.10524.x Brown DW Giles WH Greenlund KJ Croft JB Disparities in cholesterol screening: falling short of a national health objective Prev Med 2001 33 517 522 11716645 10.1006/pmed.2001.0928 Dressler DD Jacobson TA Effects of race on lipid-lowering management in hospitalized patients with coronary heart disease Am J Cardiol 2004 93 1167 1170 15110215 10.1016/j.amjcard.2004.01.052 Safford M Eaton L Hawley G Brimacombe M Rajan M Li H Pogach L Disparities in use of lipid-lowering medications among people with type 2 diabetes mellitus Arch Intern Med 2003 163 922 928 12719201 10.1001/archinte.163.8.922 Jencks SF Cuerdon T Burwen DR Fleming B Houck PM Kussmaul AE Nilasena DS Ordin DL Arday DR Quality of medical care delivered to Medicare beneficiaries: A profile at state and national levels JAMA 2000 284 1670 1676 11015797 10.1001/jama.284.13.1670 Simpson R.J.,Jr. Sueta CA Boccuzzi SJ Lulla A Biggs D Londhe A Smith S.C.,Jr. Performance assessment model for guideline-recommended pharmacotherapy in the secondary prevention of coronary artery disease and treatment of left ventricular dysfunction Am J Cardiol 1997 80 53H 56H 9372999 10.1016/S0002-9149(97)00821-7 Zeger SL Liang KY Albert PS Models for longitudinal data: a generalized estimating equation approach Biometrics 1988 44 1049 1060 3233245 Kuss O How to Use SAS for Logistic Regression of Correlated Data 2002 Paper 261-27. In: Proceedings of the Tenty-Seventh Annual SAS Users Group International Conference. Ford ES Mokdad AH Giles WH Mensah GA Serum total cholesterol concentrations and awareness, treatment, and control of hypercholesterolemia among US adults: findings from the National Health and Nutrition Examination Survey, 1999 to 2000 Circulation 2003 107 2185 2189 12719276 10.1161/01.CIR.0000066320.27195.B4 Hyman DJ Simons-Morton DG Ho K Dunn JK Rubovits DS Cholesterol-related knowledge, attitudes, and behaviors in a low-income, urban patient population Am J Prev Med 1993 9 282 289 8257617 Giles WH Anda RF Jones DH Serdula MK Merritt RK DeStefano F Recent trends in the identification and treatment of high blood cholesterol by physicians. Progress and missed opportunities JAMA 1993 269 1133 1138 8240474 10.1001/jama.269.9.1133 Thomas J Lackland D Taylor K Disparity between whites and african-americans in knowledge and treatment of cholesterol. Carolina heart survey Ann Epidemiol 2000 10 460 11018372 10.1016/S1047-2797(00)00106-X McBride P Schrott HG Plane MB Underbakke G Brown RL Primary care practice adherence to National Cholesterol Education Program guidelines for patients with coronary heart disease Archives of Internal Medicine 1998 158 1238 1244 9625403 10.1001/archinte.158.11.1238 Stafford RS Blumenthal D Pasternak RC Variations in cholesterol management practices of U.S. physicians Journal of the American College of Cardiology 1997 29 139 146 8996306 10.1016/S0735-1097(96)00441-X Chin MH Zhang JX Merrell K Diabetes in the African-American Medicare population. Morbidity, quality of care, and resource utilization Diabetes Care 1998 21 1090 1095 9653601 Probstfield JL How cost-effective are new preventive strategies for cardiovascular disease? Am J Cardiol 2003 91 22G 27G 12781905 10.1016/S0002-9149(03)00229-7 Brandle M Davidson MB Schriger DL Lorber B Herman WH Cost effectiveness of statin therapy for the primary prevention of major coronary events in individuals with type 2 diabetes Diabetes Care 2003 26 1796 1801 12766112 Kaplan GA Keil JE Socioeconomic factors and cardiovascular disease: a review of the literature Circulation 1993 88 1973 1998 8403348 Consoli SM Bruckert E Educational level has a major impact on the representations of cholesterol: a study in 1579 hypercholesterolemic patients Prev Med 2004 38 323 329 14766115 10.1016/j.ypmed.2003.10.009 Chong PH Tzallas-Pontikes PJ Seeger JD Stamos TD The low-density lipoprotein cholesterol-lowering effect of pravastatin and factors associated with achieving targeted low-density lipoprotein levels in an African-American population Pharmacotherapy 2000 20 1454 1463 11130218 10.1592/phco.20.19.1454.34855 Mottur-Pilson C Snow V Bartlett K Physician explanations for failing to comply with "best practices" Eff Clin Pract 2001 4 207 213 11685978 Wagner EH Austin BT Davis C Hindmarsh M Schaefer J Bonomi A Improving chronic illness care: translating evidence into action Health Aff (Millwood ) 2001 20 64 78 11816692 10.1377/hlthaff.20.6.64 Maviglia SM Teich JM Fiskio J Bates DW Using an electronic medical record to identify opportunities to improve compliance with cholesterol guidelines J Gen Intern Med 2001 16 531 537 11556929 10.1046/j.1525-1497.2001.016008531.x Kinn JW O'Toole MF Rowley SM Marek JC Bufalino VJ Brown AS Effectiveness of the electronic medical record in cholesterol management in patients with coronary artery disease (Virtual Lipid Clinic) Am J Cardiol 2001 88 163 5, A5 11448414 10.1016/S0002-9149(01)01612-5 Foley KA Vasey J Alexander CM Markson LE Development and validation of the hyperlipidemia: attitudes and beliefs in treatment (HABIT) survey for physicians J Gen Intern Med 2003 18 984 990 14687256 10.1111/j.1525-1497.2003.30114.x Green BL Lewis RK Wang MQ Person S Rivers B Powerlessness, destiny, and control: the influence on health behaviors of African Americans J Community Health 2004 29 15 27 14768932 10.1023/B:JOHE.0000007442.70657.75 Boulware LE Cooper LA Ratner LE LaVeist TA Powe NR Race and trust in the health care system Public Health Rep 2003 118 358 365 12815085 Cooper-Patrick L Gallo JJ Gonzales JJ Vu HT Powe NR Nelson C Ford DE Race, gender, and partnership in the patient-physician relationship JAMA 1999 282 583 589 10450723 10.1001/jama.282.6.583 Massing MW Foley KA Sueta CA Chowdhury M Biggs DP Alexander CM Simpson R.J.,Jr. Trends in lipid management among patients with coronary artery disease: has diabetes received the attention it deserves? Diabetes Care 2003 26 991 997 12663562 Sueta CA Massing MW Chowdhury M Biggs DP Simpson R.J.,Jr. Undertreatment of hyperlipidemia in patients with coronary artery disease and heart failure J Card Fail 2003 9 36 41 12612871 10.1054/jcaf.2003.5 Foley KA Massing MW Simpson R.J.,Jr. Alexander CM Markson LE Population implications of changes in lipid management in patients with coronary heart disease Am J Cardiol 2004 93 193 195 14715344 10.1016/j.amjcard.2003.09.035 Lurie N What the federal government can do about the nonmedical determinants of health. Taking a "systems" approach to structuring our government's health investments is an important first step in addressing the many contributors to health and well-being Health Aff (Millwood) 2002 21 94 106 11900189 10.1377/hlthaff.21.2.94 McGinnis MJ Williams-Russo P Knickman JR The case for more active policy attention to health promotion. To succeed, we need leadership that informs and motivates, economic incentives that encourage change, and science that moves the frontiers Health Aff (Millwood) 2002 21 78 93 11900188 10.1377/hlthaff.21.2.78 Wagner EH Chronic disease management: what will it take to improve care for chronic illness? Eff Clin Pract 1998 1 2 4 10345255

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-281532915310.1186/1471-2334-4-28Research ArticleExternal decontamination of wild leeches with hypochloric acid Aydin Atakan [email protected] Hasan [email protected] Samet Vasfi [email protected] Nezahat [email protected] Betigul [email protected] Serdar [email protected] Emre [email protected] Sinan Nur [email protected] Department of Plastic and Reconstructive Surgery, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey2 Department of Clinical Microbiology, Istanbul University, Istanbul Faculty of Medicine, Istanbul, Turkey2004 25 8 2004 4 28 28 19 1 2004 25 8 2004 Copyright © 2004 Aydin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Medicinal leech, Hirudo medicinalis, has been used in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps. In many countries, wild leeches are still provided from local markets and utilised with antibiotic prophylaxies. In this research, results of identification of bacteria in the transport fluid is reported, oral and intestinal floras and the antibiograms of the identified microorganisms are investigated. Also, to avoid possible infections, the ability of hypochloric acid, a disinfectant, to suppress the relevant microorganisms without changing the life style and behavior of leeches in terms of sucking function, is investigated. Methods Bacterial identifications and antibiograms of oral and intestinal flora and transport medium were performed for 10 leeches. The optimum concentration of hypochloric acid which eliminated microorganisms without affecting the viability and sucking function of the leeches were determined by dilution of hypochloric acid to 100, 50, 25, 12.5, 6.25 ppm concentrations in different groups of 25 leeches. Finally, 20 leeches were applied atraumatically to the bleeding areas of rats, the duration of suction was determined and compared statistically between the leeches treated and not treated with hypochloric acid solution. Results Aeromonas hydrophilia was the most commonly identified microorganism and found to be resistant to first generation cephalosporins, frequently used in prophylaxis at surgical wards. In the next stages of the study, the leeches were subjected to a series of diluted hypochloric acid solutions. Although disinfection of the transport material and suppression of the oral flora of hirudo medicinalis were successful in 100, 50, 25, 12.5, 6.25 ppm concentrations; 12.5 ppm solution was the greatest concentration in which hirudo medicinalis could survive and sucking function was not affected significantly. Conclusions External decontamination of wild leeches with 12.5 ppm hypochloric acid enables bacterial suppression without causing negative effects on leech sucking function and life. ==== Body Background The medicinal leech, Hirudo medicinalis, has been used with increasing frequency during the past few decades for salvage of venous compromised pedicled flaps, microvascular free-tissue transfers and replantations. Although the therapeutic use of leeches in medicine dates back 50 BC; for centuries they were collected from various water supplies and utilised under septic conditions with the risk of wound infection and infestation. The supply of leeches was modernized by medicinal leech farm set up in the 1970s [1]. Today, leech therapy is indicated in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps (Fig 1,2,3,4) or replants with an increase in success rate by 60%–83%. However, in the presence of infection as a complication of medicinal use of leeches; the success rate for flap salvage may decrease to ≤ 30% [2]. Aeromonas is the most common microorganism in leech infections and may cause a wide spectrum of deseases such as cellulitis, ocular infections, arthritis, myocarditis, peritonitis, meningitis, bacteremia and sepsis [2-5]. In many countries, wild leeches are still provided from local markets and utilised in many plastic and reconstructive surgery clinics depending on antibiotic prophylaxies. In the first stage of the study, bacterial content of the transport fluid of leeches that were bought from local markets were studied, also oral and intestinal flora cultures and antibiograms of the identified microorganisms were performed subsequently. In the next stages hypochloric acid, a disinfectant, was tested on animals that were not bred in specific laboratories, to suppress the relevant microorganisms in order to avoid possible infections, without changing the life style and behavior of leeches in terms of sucking function. Methods This study was approved by the Animal Care and Ethics Committee of our institution. Stage 1 Ten leeches were obtained with their 100 cc original water from different stores. Their sizes varied between 6–10 cm in length and 0, 5 – 1, 5 cm in width. They were transported in sterile boxes immersed in the water which the animals were kept at the market. For 2 days they were observed at room temperature. Specimens were taken and smears from transport fluids were prepared. All leeches were held with sterile gloves and prepared in sterile conditions. Specimens were obtained from their mouth (the smaller and more mobile pole) with the help of sterile cotton – swab. After traction was applied between the mouth and posterior muscular organ, the animal was cleaned with alcoholic solution of povidone-iodine, as described by Hokelek et al. [6]. A longitudinal incision was made on the animal and crop or intestine was exposed (Figure 5). Using sterile cotton-swabs, smears were prepared from the intestinal material and they were planted in MacConkey and blood agars in 5 minutes. The growth was observed after 24–48 hours of incubation at 37°C. In addition to classical bacterial identification methods, Analytical Profile Index 32 GN (BioMérieux, France) kit was also used for identification. Further biochemical studies were made on microorganisms that were found to be A. hydro/caviae and A. sobria for definite identification. Since bacterial growth was observed in specimens taken from oral and intestinal flora or transport fluid of all animals except one ; antibiograms for aeromonas isolates were performed. Stage2 30 leeches from different stores were obtained and transported in different sterile boxes with their 100 cc original water as in stage 1. On the following day, samples were taken from transport fluids and oral region and planted in MacConkey and blood agar. Twenty five leeches, which had bacterial growth in either oral flora or transport water were used for the second stage. 100, 50, 25, 12.5, 6.25 ppm solutions were prepared by adding apropriate amounts of hypochloric acid into the transport fluids of 25 leeches seperately with five animals in each concentration group. After 10 minutes of contact with hypochloric acid solutions at 20°C, smears were taken from mouths and transport fluids of each leech under sterile conditions with cotton-swabs. Cotton-swabs were put into a neutralizing media (lecithinized agar media) because they could also contain disinfectant fluid which would prevent microorganism reproduction. Then the neutralized smears were planted into MacConkey and blood agar [7]. The leeches were then put into boxes containing distilled water and observed. Although the leeches which had been left in the 100, 50, 25 ppm solutions lost their muscular activity and died ;the leeches which were treated with 12.5 and 6.25 ppm hypochloric acid solutions were alive. Bacterial growth was not observed in either concentration group for the specimens taken from oral flora and transport fluid, however bacterial cultures were positive for in specimens taken from the intestine or crop. Of these microorganisms, bacterial colonies for Aeromonas spp were counted. To compare the changes in the number of Aeromonas colonies in leech intestinal flora induced with the application of hypochloric acid, the intestine of five new leeches were exposed as described for Stage 1. Based on the findings of Stage 2, a concentration of 12.5 ppm was chosen for Stage 3 studies. Stage 3 20 leeches were obtained with their 100 cc original water from different stores and equally divided into two groups. One group was treated with hypochloric acid while the others had no treatment at all. After taking specimens from the transport fluids and oral floras, for 10 leeches 12.5 ppm hypochloric acid solution was prepared with the transport fluids. After waiting for 10 min at 20°C temperature, smears were taken from the oral floras and fluids again. All leeches were put into boxes containing distilled water and watched for survival and blood suction function on sedated rats (ketamine 30 mg/kg and xylazine 10 mg/kg). After shaving dorsal region of 20 rats, 2 mm long skin incissions were done in order to produce bleeding. The leeches were applied to the bleeding areas atraumatically and the duration of suction was determined and compared statistically (ANOVA) between the leeches treated and not treated with hypochloric acid solution. Statistical significance was presumed at p < 0.05. Results Stage 1 Bacterial growth was observed in specimens taken from the oral flora in 4 animals (40%), intestinal flora in 9 animals (90%) and transport media in 5 animals (50%). In one leech, no growth was observed in either oral, intestinal and transport fluid cultures. In all culture media, 4 different gram (-) bacterial growth (aeromonas species, peudomonas species, acinetobacter species, sphingobacterium species) was observed. In one media, Gram (+) growth was seen :meticilline susceptible coagulase (-) staphylococcus. Dominant microorganisms were aeromonas species, Table 1. This was an expected result. The biochemical characterization and identification of Aeromonas spp. was investigated by using conventional methods and a commercial kit ID 32 GN ATB System (BioMérieux). In this study, with this system the results were obtained as A. hydro/caviae, A. sobria, A. salmonicidia and A. spp, with the latter nomenclature designating all other Aeromonas species. The most commonly encountered agents, A. hydro/caviae and A. sobria underwent further biochemical studies, as suggested by Abbott, et al.[4]. As a result, it was seen that A. hydro/caviae was in fact A. hydrophila, and A. sobria was in fact A. veronii biovar sobria (Table 2) Antimicrobial susceptibility testing was performed by the disc diffusion method according to NCCLS (National Commity Clinical Laboratory Standart / American). The Aeromonas spp. were found to be susceptible to third generation cephalosporines, aminoglycosides and co-trimoxazole, and all aeromonas spp. were resistant to ampicillin-sulbactam and amoxycillin-clavulanic acid (Table 3). Three Aeoromonas spp. were found to have inducable beta lactamase which meant that although they were susceptible, they might develop resistance to beta lactams other than carbapenems during treatment. Stage 2 Fluid and oral flora cultures that were prepared before hypochloric acid application showed multiple microorganism growth, as it was in stage 1. After adding hypochloric acid there was no growth in transport fluid and oral media cultures in either concentration so that no identification and antibiogram study could be performed. After 10 minutes in hypochloric acid solution, leeches were taken to sterile containers which contained distilled water. The muscle activity of all leeches that had been treated in 100 ppm solution ended in 3 min, in 50 ppm ended in 7 min, in 25 ppm ended in 21 min and they died eventually. The ones which had been immersed in 12.5 ppm and 6.25 ppm solutions survived. The Aeromonas colonies in the intestinal flora of these last two groups were compared with the control animals'. With respect to colony suppression, while the difference between the control group, 12.5 ppm group was significant, (p < 0.05, F = 30.1, ANOVA) however the difference between control group and 6.25 ppm group was not significant. Stage 3 All the leeches survived while hyperactivity followed by hypoactivity was seen in the group of leeches treated with hypochloric acid. In this group, two of the leeches never made suction but the other eight leeches sucked the bleeding area for 5–16 min (mean 12.2 ± 3.8). Non hypochloric acid treated group sucked for 9–22 min (mean 15.5 ± 4.4) and the difference between two groups was not significant (p > 0.05, F = 2.6, ANOVA). In 70 % of cultures that were prepared from transport fluids of leeches and in 50% of cultures that were prepared from oral floras of leeches before hypochloric acid application, bacterial growth (mainly Aeromonas spp) were observed. After hypochloric acid application no growth was observed in any of the specimens taken from animals, therefore no identifications or antibiograms could be performed. Discussion Many reports showed that venous compromise is a more common complication than arterial occlusion in free-tissue transfers. Although surgical correction is the first choice for management of venous compromised flaps ; in cases where surgical correction is not feasible or fails, nonsurgical procedures such as exanguination treatments can be used. Among them, medicinal leech therapy has become widely accepted to promote perfusion of venous compromised flaps and to relieve congestion. However there are reports in the literature concerning about infections specially with aeromonas bacteria after leech therapy calling attention for appropriate antibiotic prophylaxis [8,9]. Many species of Aeromonas survive in water. These also can live in materials embedded in water, drainage tubes, fountains and containers for distilled water. Among other aeromonas species A. hydrophilia is the mostly isolated one from the intestines of leeches. Hemoglobin that is sucked by the leech is denaturated by aeromonas. One of the products, heme, is used by aeromonas and the other, globulin, is used by the leech[10]. This shows the endosymbiotic relationship of leeches and aeromonas. Many investigators isolated aeromonas spp as the dominant bacteria in infections related with leech usage [11-13]. Aeromonas spp. can cause infections with contamination through the bite point of the leech or on the macerated skin. The infections caused by these microorganisms can be minimized by easy measures. Leeches should not be handled with the forceps from the containers because these traumas can cause regurgitation of the leech into the wound and eventual contamination. Therefore, leeches should be taken with sterilized gloves without traumatisation of the animal. Same care should be taken while carrying the leech from the wound. When the leech is filled up, it puts itself off. If it is taken before this time, the risk of regurgitation is high. Low amounts of cigarette smoke or a heat source can be used in order to cause the leech to put itself off. During leech application, the routine surgical antibiotic prophylaxis must be changed because aeromonas spp. are resistant to first generation cephalosporins which are widely used at surgical wards. These are usually susceptible to third generation cephalosporins, aminoglycosides, tetracyclines and quinolones. Lineaweaver et al. recommended third generation cephalosporins for the prophylaxis of surgical procedures after which leeches were utilised [10,12]. In countries, where special farms for medicinal leeches do not exist, animals obtained from markets should be studied for their floras and antibiotic susceptibilities. Eroğlu et al. investigated the floras and the antibiotic susceptibility of leeches in Black Sea region. Most commonly A. hydrophila, Ochrobacter antropia, nonfermantating gram (-) rods, Acinetobacter Iwofii and A sobria were isolated. All of the isolates were sensitive to ciprofloxacine, cefotaxime, ceftazidime, gentamycine and trimetoprim-sulfamethoxasole [14]. Likewise, we aimed to investigate the general flora of leeches we use occasionally and antibiotic susceptibility of these bacteria. Our results correlated with the other studies about this subject in the literature. The interesting observation in our study was there were 3 Aeromonas hydrophylia colonies which produced inducable betalactamase. In case of infections with these species, beta lactam antibiotics except carbapenems should not be used. In addition, we aimed to supress possible bacterial contamination of the transport media and oral floras of leeches but not intestinal flora because of the endosymbiotic relationship of the aeromonas and leech, with hypochloric acid solutions at disinfectant concentrations that does not affect the life style of leeches. Investigators have attempted to disinfect the guts of leeches before they are placed on patients by placing the ectoparasites in % 0.02 chlorhexidine for 15 seconds or in antibiotic solutions (tetracycline or cefoperazone solutions) for 12 hours, but these attempts were unsuccessful [2]. Mackay et al. incubated leeches in solutions of antibiotics to which Aeromonas spp. are sensitive for 12 hours but could not eradicate aeromonas from the intestines [11]. We preferred hypochloric acid which is an ideal disinfectant and has a wide antibacterial spectrum for desired purpose. Chlorine in the form of hypochloric acid exhibits rapid microbicidal activity by inhibiting of key enzymatic reactions within the cell and eventual protein denaturation. It has a rapid bactericidal effect and can dissolve in water. However, it has some disadvantages as it irritates mucosal membranes, has interactions with some chemicals and metals [15]. It was showed that bactericidal effect of hypochloric acid of 100 ppm concentrations on pseudomonas was achieved within 10 min exposure [7]. Aeromonas spp. cause similar nosocomial infections and has similar antibiotic susceptibility with pseudomonas spp. Therefore, in the second stage of our study, beginning with 100 ppm, we applied decreasing concentrations of hypochloric acid in transportation fluids, observed bacterial growth by taking specimens from transport material and oral flora of hirudo medicinalis. We tried to find the dilution ratio that was closest to 100 ppm, suppresing oral flora and transfer liquid and also allowing the leech to survive with normal function. As a result of the second stage of the study we found out that concentration to be 12.5 ppm. In the third stage, we observed that mean sucking duration of the hypochloric acid treated group was shorter than the other group but the difference was not significant (12.2 min & 15.5 min respectively). However hypochloric acid had successful disinfection effect for transport liquid and oral flora. In our opinion, such a decrease in duration of sucking function can be preferable to infection possibility. Since leeches are much cheaper than antibiotics, it is logical to use them once, after treating with hypochloric acid, and never utilise them again. We do not advise to use "full-up" leeches over and over again by putting them in hypertonic solutions to force them vomitting because if they regurgitate, the intestinal flora can readly contaminate the environment where the leeches are kept and cause infection eventually. Although the oral flora and transport enviroment studies do not really address the problem since excretory contamination is probably the major factor in leech infections ; we believe that to take any measure in order to decrease the infection risk during utilisation of ordinary leeches is valuable. Conclusions We can comment that preparation of 12.5 ppm hypochloric acid solution with transport fluids of ordinary leeches obtained from the local market for 10 minutes and then taking the leeches gently from water before application can prevent possible infections caused by contamination from leech oral flora and transport medium. Competing interest None declared. Author's contributions AA and SVK participated in design of the study, involved in the dissections of leeches. EH and ST participated in the design of the study and writing of the manuscript.HN, NG and BO involved in the antibiogram tests. SNK participated in design of the study. Pre-publication history The pre-publication history for this paper can be accessed here: Figures and Tables Figure 1 Tissue defect on the dorsum of the hand of a male patient after a crush type injury. Figure 2 Venous insufficiency of the posterior interosseous flap used for defect reconstruction. Figure 3 Leech usage in the treatment of venous insufficiency. Figure 4 Late follow up of the salvaged flap. Figure 5 Intestinal exploration of Hirudo medicinalis and taking specimen with cotton swab. Table 1 Results of the cultures prepared from transport fluids, oral and intestinal smears of 10 leeches. Number of leech Source of the culture material Bacterial Growth 1 oral flora no reproduction intestinal flora no reproduction transport fluid no reproduction 2 oral flora Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp intestinal flora Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp transport fluid Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp 3 oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid no reproduction 4 oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp 5 oral flora no reproduction intestinal flora Aeromonas sobria transport fluid no reproduction 6 oral flora no reproduction intestinal flora Aeromonas sobria transport fluid Acinetobacter spp, methicillin sensitive-coagulase negative Staphylococcus spp 7 oral flora Aeromonas salmonicidia, Pseudomonas spp, Sphingobacterium spp intestinal flora Aeromonas hydro/cavie, Aeromonas sobria transport fluid Aeromonas hydro/cavie, Aeromonas salmonicidia 8 oral flora Aeromonas hydro/cavie intestinal flora Aeromonas sobria transport fluid Pseudomonas spp 9 oral flora Aeromonas hydro/cavie intestinal flora Aeromonas hydro/cavie transport fluid no reproduction 10 oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid no reproduction Table 2 Definitive identification of A. hydro/caviae and A. sobria with biochemical tests. No 2 3 4 5 6 7 8 9 10 ◆ O I F I I F I I I F O I O I I Results of API 32 GN h/c h/c h/c h/c h/c h/c s s h/c s h/c h/c s h/c h/c h/c M + + + + + + + + + + + + + + + + Esculin + + + + + + - - + - + + - + + + Catalase + + + + + + + + + + + + + + + + Indole + + + + + + + + + + + + + + + + VP + + + + + + + - + + + + + + + + LDC + + + + + + + + + + + + + + + + ODC - - - - - - - - - - - - - - - - DNase + + + + + + + + + + + + + + + + Hem. + + + + + + + + + + + + + + + + Glucose + + + + + + + + + + + + + + + + Lactose - - - - - - - - - - - - - - - - Urea - - - - - - - - - - - - - - - - Citrate + + + - + + - - - + - + + + + - Results h h h h h h v v h v h h v h h h Abbreviations : No: Number of leech, ◆: Source of the culture material, API 32 GN: Analitycal Profile Index 32 GN (BioMérieux), O: Oral flora, I: Intestinal flora, F: Transport fluid, h/c: A. hydro/caviae, h: A. hydrophila, s: A. sobria, v: A. veronii biogroup sobria, M: Motility, VP: Voges-Proskauer, LDC: Lysine decarboxylase, ODC: Ornithine decarboxylase, Hem: Hemolysis, Lactose: Asit from Lactose. Table 3 The antibiograms of aeromonas isolates from 9 different leeches. Leech Antibiotics 1st 2nd 3rd 4th 5th 6th 7th 8th 9th h h h v v h h h h AMP R R R R R R R R R SAM R R R R R R R R R AMC R R R R R R R R R CZ R R R S S R R R S CXM S S S S S S S S S FOX S S S S S S S S S CRO S S S S S S S S S SCF S S S S S S S S S FEP S S S S S S S S S GM S S S S S S S S S TOB S S S S S S S S S NET S S S S S S S S S AK S S S S S S S S S SXT S S S S S S S S S NOR S S S S S S S S S IPM S S S S S S S S S MEM S S S S S S S S S TZP S S S S S S S S S AZM S S S S S S S S S IBL + + + Abbreviations : h:A. hydrophila, v:A. veronii biogroup sobria, R:Resistant, S:Sensitive AMP:Ampicillin, SAM:Sulbactam/ampicillin, AMC:Amoxicillin/clavulanate, CZ:Cefazolin, CXM:Cefuroxim FOX:Cefoxitin, CRO:Ceftriaxone, SCF:Sulbactam/cefoperazone, FEP:Cefepime, GM:Gentamicin, TOB:Tobramycin, NET:Netilmicin, AK:Amikacin, SXT:Trimethoprim/sulfamethoxazole, NOR:Norfloxasin, IPM:Imipenem, MEM:Meropenem, TZP:Piperacillin/tazobactam, AZM:Aztreonam, Table 4 The number of bacterial colonies before and after hypochloric acid applications of 12.5 ppm and 6.25 ppm concentrations and control group. No Concentration of hypochloric acid Transport fluid Colony count Mouth Colony count Gut Colony count 1 before 12 16 Not studied 12.5 ppm after No reproduction No reproduction 25 2 before 23 14 Not studied 12.5 ppm after No reproduction No reproduction 18 3 before 30 34 Not studied 12.5 ppm after No reproduction No reproduction 50 4 before 25 16 Not studied 12.5 ppm after No reproduction No reproduction 44 5 before 18 No reproduction Not studied 12.5 ppm after No reproduction No reproduction 30 1 before No reproduction 20 Not studied 6.25 ppm after No reproduction No reproduction 100 2 before 27 7 Not studied 6.25 ppm after No reproduction No reproduction 120 3 before 20 No reproduction Not studied 6.25 ppm after No reproduction No reproduction 72 4 before No reproduction 8 Not studied 6.25 ppm after No reproduction No reproduction 95 5 before 15 12 Not studied 6.25 ppm after No reproduction No reproduction 75 1 Control group Not studied Not studied 100 2 Not studied Not studied 150 3 Not studied Not studied 120 4 Not studied Not studied 105 5 Not studied Not studied 140 ==== Refs Rao J Whitaker IS Use of Hirudo medicinalis by maxillofacial surgical units in the United Kingdom: current views and practise British Journal of Oral and Maxillofacial Surgery 2003 41 54 55 10.1016/S0266-4356(02)00284-X Sartor C Limouzin PL Legre R Casanova D Bongrand MC Sambuc R Drancourt M Nosocomial infections with Aeromonas hydrophila from leeches Clinical Infectious Diseases 2002 35 1 5 12060868 10.1086/340711 Fenollar F Fournier PE Legre R Unusual case of Aeromonas sobria cellulitis associated with the use of leeches European Journal of Clinical Microbiology and Infectious Diseases 1999 18 72 73 10.1007/s100960050232 Abbott SL Cheung WKW Janda JM The Genus Aeromonas: Biochemical Characteristics, Atypical Reactions, and Phenotypic Identification Schemes Journal of Clinical Microbiology 2003 41 2348 2356 12791848 10.1128/JCM.41.6.2348-2357.2003 Ouderkirk JP Bekhor D Turett GS Murali R Aeromonas Complicating Medicinal Leech Therapy Clinical Infectious Diseases 2004 38 36 37 10.1086/381438 Hokelek M Guneren E Eroglu C An experimental study to sterilize medicinal leeches European Journal of Plastic Surgery 2002 25 81 85 10.1007/s00238-002-0348-x Klingeren BV Pullen W Comparative testing of disinfectants against Mycobacterium tuberculosis and Mycobacterium terrae in a quantitative suspension test Journal of Hospital Infection 1987 10 292 298 2891759 10.1016/0195-6701(87)90012-0 Kubo T Yano K Hosokawa K Management of flaps with compromised venous outflow in head and neck microsurgical reconstruction Microsurgery 2002 22 391 395 12497578 10.1002/micr.10059 Utley DS Koch RJ Goode RL The failing flap in Facial Plastic and Reconstructive Surgery:role of the medicinal leech Laryngoscope 1998 108 1129 1135 9707230 10.1097/00005537-199808000-00005 Richerson JT Davis JA Meystrik R Aeromonas, acclimation, and penicillin as complications when leeches are applied to skin flaps in rabbits Laboratory Animals 1990 24 147 150 2366512 Mackay DR Manders EK Saggers GC Banducci DR Prinsloo J Klugman K Aeromonas species isolated from medicinal leeches Annals of Plastic Surgery 2000 45 568 569 Lineaweaver WC Hill MK Buncke GM Follansbee S Buncke HJ Wong RK Manders EK Grotting JC Anthony J Mathes SJ Aeromonas hydrophila infections following use of medicinal leeches in replantation and flap surgery Annals of Plastic Surgery 1992 29 238 244 1524373 Graf J Symbiosis of Aeromonas veronii Biovar sobria and Hirudo medicinalis, the Medicinal Leech: a Novel Model for Digestive Tract Associations Infection and Immunity 1999 67 1 7 9864188 Eroglu C Hokelek M Guneren E Esen S Pekbay A Uysal OA Bacterial flora of hirudo medicinalis and their antibiotic sensitivities in the Middle Black Sea Region Annals of Plastic Surgery 2001 47 70 73 11756807 10.1097/00000637-200107000-00013 William AR David JW Uses of inorganic hypochloride(bleach)in health-care facilities Clinical Microbiology Reviews 1997 10 597 610 9336664

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==== Front BMC Pregnancy ChildbirthBMC Pregnancy and Childbirth1471-2393BioMed Central London 1471-2393-4-181533313810.1186/1471-2393-4-18Research ArticleMalaria and anemia prevention in pregnant women of rural Burkina Faso Miaffo Caroline [email protected] Florent [email protected] Bocar [email protected] Albrecht [email protected] Olaf [email protected] Ministry of Public Health, POB 2997, Yaoundé, Cameroon2 Centre de Recherche en Santé de Nouna, POB 02, Nouna, Burkina Faso3 Department of Tropical Hygiene and Public Health, Ruprecht-Karls-University, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany2004 27 8 2004 4 18 18 23 4 2004 27 8 2004 Copyright © 2004 Miaffo et al; licensee BioMed Central Ltd.2004Miaffo et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Pregnant women are a major risk group for malaria in endemic areas. Only little information exists on the compliance of pregnant women with malaria and anaemia preventive drug regimens in the rural areas of sub-Saharan Africa (SSA). In this study, we collected information on malaria and anaemia prevention behaviour in pregnant women of rural Burkina Faso. Methods Cross-sectional qualitative and quantitative survey among 225 women of eight villages in rural northwestern Burkina Faso. Four of the villages had a health centre offering antenatal care (ANC) services while the other four were more than five kilometers away from a health centre. Results Overall ANC coverage (at least one visit) was 71% (95% in health centre villages vs 50% in remote villages). Malaria and anaemia were considered as the biggest problems during pregnancy in this community. ANC using women were quite satisfied with the quality of services, and compliance with malaria and anaemia prevention regimens (chloroquine and iron/folic acid) was high in this population. Knowledge on the benefit of bed nets and good nutrition was less prominent. Distance, lack of money and ignorance were the main reasons for women to not attend ANC services. Conclusions There is an urgent need to improve access of rural SSA women to ANC services, either through increasing the number of rural health centres or establishing functioning outreach services. Moreover, alternative malaria and anaemia prevention programmes such as intermittent preventive treatment with effective antimalarials and the distribution of insecticide-treated bed nets need to become implemented on a large scale. ==== Body Background Each year between 75.000 and 200.000 infant deaths are attributed to malaria infection in pregnancy globally, and between 200.000 and 500.000 pregnant women develop severe anaemia as a result of malaria in Sub-Saharan Africa (SSA) [1]. Pregnant compared to non-pregnant women are at an increased risk for malaria, and the severity of the clinical manifestations in the women and her foetus depends on the level of pre-pregnancy immunity [2]. While in areas of low malaria endemicity all pregnant women are equally susceptible to the consequences of malaria infection, in areas of high endemicity women appear to be most susceptible during their first pregnancy [3]. However, more recent publications point to significant susceptibility in primigravidae as well as in multigravidae [4]. Pregnancies in women living in malaria endemic regions are associated with a high frequency and density of P. falciparum parasitaemia, with high rates of maternal morbidity including fever and severe anaemia, with abortion and stillbirth, and with high rates of placental malaria and consequently low birth weight in newborns caused by both prematurity and intrauterine growth retardation [1,3,5]. In order to reduce malaria-related ill health, regular chemoprophylaxis has been recommended to all pregnant women living in malaria-endemic areas [6]. Most African countries, including Burkina Faso, include routine chemoprophylaxis in their official antenatal care programmes. However, in practice, coverage of chemoprophylaxis is limited due to low accessibility and quality of antenatal care (ANC) services as well as problems with compliance [7]. It has been estimated from a survey in four African countries that less than 20% of women use a prophylactic regimen close to the WHO recommendations [8]. While insecticide-treated bed nets and curtains (ITN) have been shown to substantially reduce malaria morbidity and mortality in children, results from initial trials on the efficacy of ITNs for malaria prevention in pregnancy produced conflicting evidence and were classified as inconclusive by the Cochrane Collaboration [3,9]. However, with the publication of the findings from a major ITN trial in a holoendemic area of western Kenya, the use of ITN during pregnancy is getting more credibility [10]. In Burkina Faso, the official policy for malaria and anaemia prevention in pregnant women comprises of chloroquine and iron/folic acid supplementation respectively. However, in the rural areas of Burkina Faso little information exists on coverage with ANC and on compliance with preventive regimens. We assessed the coverage of antenatal care and investigated the knowledge and adoption of preventive practices with respect to malaria and anaemia in users and non-users of antenatal care. Methods Study area The study took place in the rural part of the research zone of the Centre de Recherche en Santé de Nouna (CRSN) in Nouna Health District, northwestern Burkina Faso. The CRSN research zone consists of Nouna town and 41 of the surrounding villages with a total population of around 60.000 inhabitants. The Nouna area is a dry orchard savanna, populated mainly by subsistance farmers of different ethnic groups. Malaria is holoendemic but highly seasonal in this part of Westafrica [11]. Most malaria transmission takes place during or shortly after the rainy season which usually lasts from June until October [11]. Modern health services in the CRSN research zone are limited to four village-based health centres and the district hospital in Nouna town. As a consequence, malaria control is mainly based on home treatment with chloroquine, the official first-line treatment drug in Burkina Faso. Roughly half of all households in the area possess at least one untreated bed net and since 2000, ITN are distributed as part of an ongoing trial in young children [12,13]. The official policy for malaria and anaemia prevention during pregnancy in Burkina Faso consists of a curative dose of 1 500 mg chloroquine during three days followed by a weekly dose of 300 mg chloroquine and a combined dose of daily 200 mg iron and 0.25 mg folic acid. This regimen should be followed from the first ANC visit until six weeks after delivery. Since the year 2002 it is the official policy in Burkina Faso to offer ANC services free of charge. This includes ANC card, physical examination and counselling, and malaria/anaemia prevention drugs. However, urine examination, gloves, and drugs for other concomitant diseases still have to be paid for. Urine examination and gloves usually cost 150–200 F CFA (1 Euro = 650 F CFA). Study design The study was cross-sectional and descriptive in nature, using both qualitative and quantitative methods for data collection. The study was implemented in May and June 2003. The research team comprised of the investigators and six trained local interviewers familiar with the common spoken local languages and French. The questionnaires were pre-tested before administration. The study took place in eight of the 41 villages of the CRSN study area. Villages were selected as follows: At a first stage, the four villages of the CRSN study area where a health centre exists were purposely selected. To account for the socio-demographic variability and geographical accessibility, in each of these health centre defined sub-areas, another village distant of at least 5 kilometres to the health centre was randomly selected. Qualitative research We conducted six Focus Group Discussions (FGD). Two with pregnant women users of ANC, two with husbands of pregnant women users of ANC, and two with pregnant women non-users of ANC. Respective FGDs were held with groups of six to 12 participants from study villages with and without a health centre. Key informant interviews were conducted with four maternity health workers, seven traditional birth attendants and 29 women group leaders. The interviews assessed their knowledge, attitudes and practices about malaria and anaemia prevention in pregnancy. Quantitative research The design of the quantitative survey instrument was informed by the results of the qualitative interviews. A structured questionnaire was administered to all women from the eight study villages who had delivered a life child during the last six months (n = 225). Information on births was available through the existing Demographic Surveillance System (DSS) in the study area [14]. The questions focussed on socio-demographic characteristics, obstetrical history, knowledge and practice of preventive measures against malaria and anaemia during pregnancy, factors influencing the utilisation of ANC services and on the compliance with chloroquine and iron/folic acid supplementation during pregnancy. The questionnaires were filled in by the interviewers who also cross-checked given answers with all available ANC cards (n = 156/225). On the ANC cards, the estimated age of pregnancy at first visit was reported through the fundal height in centimetres, as women were not able to recall their last menstruation period during ANC visits. These data were afterwards transformed into a specific scoring system. We used two definitions for malaria prophylaxis. A complete curative dose of 1 500 mg chloroquine followed by regularly weekly 300 mg doses afterwards (complete prophylaxis), and an incomplete regimen consisting of only 300 mg weekly doses (incomplete prophylaxis). A combined dose of daily 200 mg iron and 0.25 mg folic acid was defined as a complete prophylaxis. Statistical analysis The data were entered in Microsoft Access 2000, cleaned, and then analysed with Epi Info 2000. Univariate analysis was done with chi-square test or Fisher's exact test to compare proportions for categorical variables. Results were considered to be significant when the 2-sided P value was <0.05. Ethical aspects We received ethical approval from the institutional Ethical Committee at the Department of Tropical Hygiene in Heidelberg, Germany, and the local Ethical Committee in Nouna, Burkina Faso. Oral informed consent was obtained from all participants. Results Study population The characteristics of participants on the quantitative survey are shown in table 1. The great majority of the survey women were married, illiterate, housewife/farmers and their ages range between 15 and 49 years. The distribution of ethnicity among survey women was as follows: 41% Bwaba, 39% Marka, 15% Mossi, 3% Samo and 2% others. The median number of pregnancies among survey women was 6 (range 1–13). There were no differences between the background characteristics of users compared to non-users of ANC services, except regarding distance to the nearest ANC services. When comparing women living at a distance ≤ 5 km with women living >5 km, distance was significantly associated with ANC use (p < 0.001). Table 1 Background characteristics of women interviewed ANC Background Characteristics (%) Users n = 159 Non Users n = 66 All n = 225 Age Group  15–19 24 (15) 12 (18) 36 (16)  20–29 81 (51) 37 (56) 118 (52)  30–49 54 (34) 17 (26) 71 (32) Education  No Schooling 145 (91) 62 (94) 207 (92)  Primary Education 14 (9) 4 (6) 18 (8) Parity  1 26 (16) 8 (12) 34 (15)  2–3 46 (29) 17 (26) 63 (28)  4–13 87 (55) 41 (62) 128 (57) Distance*(Km)  0–4 99 (62) 5 (8) 104 (46)  5–7 43 (27) 59 (89) 102 (45)  8–15 17 (11) 2 (3) 19 (8) * Distance to the nearest health center in kilometers ANC coverage In this study population, the minimal ANC coverage (defined as at least one ANC visit during pregnancy) was 159/225 (71%) and 63/225 (28%) if we consider the optimal frequency of at least three ANC visits (national goal). Minimal ANC coverage was 97/102 (95%) in villages with a health centre vs 62/123 (50%) in remote villages (p < 0.001), while the optimal ANC coverage was 55/102 (54%) in villages with a health centre vs 8/123 (7%) in remote villages (p < 0.001). Among ANC users, 27%, 40% and 33% of women visited ANC services one time, two times or more than two times respectively during their pregnancy. The first ANC visit of ANC users was in 14% during the first trimester, in 57% during the second trimester, and in 27% during the third trimester. Malaria and anaemia prevention knowledge and behaviour Malaria and anaemia were considered as the most common diseases during pregnancy by the majority of the participants in the FGD, key informant interviews and survey women. In the Dioula language (lingua franca in the region) malaria is equivalent to "Soumaya", light to moderate anaemia to "Djolidessé" and severe anaemia to "Djoliban". Most women in the FGD were knowledgeable about the malaria prevention effect of chloroquine and the anaemia prevention effect of iron/folic acid. The knowledge of malaria and anaemia prevention measures by ANC users is given in table 2. Regarding malaria, the majority of survey women stated that it can be prevented with chloroquine (white tablets), while only a minority mentioned mosquito nets or others measures. Regarding anaemia, iron/folic acid (red colour tablets or vitamins) supplementation was stated by the majority of survey women as being protective, while a much smaller percentage of women mentioned nutrition as an important factor. Stating chloroquine and mosquito nets as prevention measures against malaria and iron/folic acid against anaemia was significantly associated with ANC use (p < 0.002; p < 0.001; p < 0.001). Table 2 Knowledge of preventive measures against malaria and anemia ANC All P Value Knowledge factors (%) Users n = 159 Non Users n = 66 All n = 225 p-value Malaria prevention  Chloroquine 128 (66) 35 (59) 163 (65) <0.001  Mosquito nets 40 (21) 2 (3) 42 (17) < 0.001  Hygiene 6 (3) 3 (5) 9 (4) n.s.  Protective clothing 12 (6) 0 (0) 12 (5) <0.05  Does not know 7 (4) 19 (32) 26 (10) <0.001 Anemia prevention  Iron/Folic acid 129 (76) 16 (25) 145 (63) <0.001  Adequate nutrition 29 (17) 8 (13) 37 (16) n.s.  Does not know 11 (7) 39 (62) 50 (22) <0.001 Table 3 shows data on self-reported use of malaria and anaemia prophylaxis with chloroquine and iron/folic acid in the population of ANC users together with respective data taken from their ANC cards. A correct prescription of chloroquine (complete prophylaxis) on ANC cards was seen in 60%, and a correct prescription of iron/folic acid (complete prophylaxis) was seen in 78%. In contrast, self-reported oral information during ANC visits on complete and incomplete prophylaxis regimens were lower and matched well with self-reported information on the chloroquine and iron/folic acid dosages taken. Most women reported being compliant with the oral information on chloroquine and iron/folic acid regimens from first ANC visit until delivery. Table 3 Prescription in ANC card of chloroquine prophylaxis and iron/folic acid supplemen-tation, self-reported ANC instructions and self-reported intake among ANC users (n = 159) Chloroquine (%) Iron/folic acid (%) Prescription on ANC card  Complete prophylaxis 95 (60) 124 (78)  Incomplete prophylaxis 29 (18) -  Incorrect prescription 32 (20) 32 (20)  No prescription (no ANC card) 3 (2) 3 (2) Instructions given at ANC visits (self-reported)  Complete prophylaxis explained 46 (29) 111 (70)  Incomplete prophylaxis explained 65 (41) -  Incorrect instructions given 41 (26) 41 (26)  No instructions given 7 (4) 7 (4) Dosages taken (self-reported)  Complete prophylaxis 46 (29) 149 (94)  Incomplete prophylaxis 67 (42) -  Incorrect dose 43 (27) 8 (5)  No prophylaxis 3 (2) 2 (1) Duration of chloroquine and iron/folic acid prophylaxis  From 1st ANC until before delivery 20 (13) 20 (13)  From 1st ANC until delivery 115 (72) 124 (78)  From 1st ANC until after delivery 21 (13) 13 (8)  No prophylaxis 3 (2) 2 (1) Factors influencing the use of ANC services Of ANC users, the great majority reported to be satisfied with the quality of ANC services. Only 16% of ANC users were aware of the fact that ANC services have recently become free of charge, and 42% reported that they still had paid for services. Most (73%) ANC users had paid between zero and 200 F CFA, while 12% and 6% had paid between 200 and 1.500 and between 1.500 and 7.500 F CFA respectively. Apart from distance to the next health centre, lack of resources and ignorance were the most frequent stated reasons during qualitative and quantitative interviews why women did not attend ANC services. The majority of ANC non-users reported no specific prophylaxis during their pregnancy, but 6% took irregularly self medication for malaria/anaemia prevention. Moreover, 30% of ANC non-users had sought advice from traditional birth attendants (TBA). Typical statements in the FGDs with non-users of ANC services werecollected as follow: • "ANC is not free of charge, you must pay for ANC card and the medicine and it could be up to 500 CFA and for me I find it is expensive. Our husbands find it expensive too, that is why the majority of us can not attend ANC services". • "I don't know the advantages of ANC services, some of the pregnant women if they don't fell sick they wouldn't accept to attend ANC ". • "There is lack of advices from health workers, if you go to the ANC for the 1sttime, they give drugs, they don't tell you when you should come back and how many visits you should attend. That is how they do here". • "If you are not sick, you don't pay for drugs, what you pay for is the ANC card at 150 F CFA...." • " But even the ANC card is free of charge, only the gloves cost 100 F CFA...." • "I had not yet heard about the free ANC services ... Since I know now that ANC is free of charge I will attend the services." • "We do not have money to go to the health centre it will be nice to have one in our village". Itching, vomiting and fatigue were the most frequently stated side effects of malaria/anaemia prevention drug regimens during interviews, sometimes leading to non-compliance. Most women also stated that bed nets are considered too expensive for their household. Discussion The main findings of this study are (1) that coverage of antenatal care is far from complete, particularly in villages without a health centre, (2) that malaria- and anaemia-related knowledge and compliance with preventive measures is comparatively high with a wide gap between users and non-users of antenatal care and (3) that health services need to improve their response to the women's need for preventive care in pregnancy. Use and coverage of antenatal care In this community-based study from a rural area of Burkina Faso, we found an overall ANC coverage of 71%, which is however not representative given the used methodology. Most women had two ANC visits during their pregnancy, mainly during the second and third trimester. As we found ANC coverage to be much higher in villages with a health centre compared to villages quite distant from a health centre, and as we included similar numbers of health centre and non-health centre villages, our ANC coverage figure is likely to be an overestimate. The national demographic and health survey in Burkina Faso claims an ANC attendance (at least one ANC during one pregnancy) of 59% [15]. Although health workers from rural health centres in Burkina Faso are advised to do regularly outreach work in the villages of their respective catchment areas, in practice such visits are rare due to a number of reasons such as lack of transport. Our findings thus support the need for better access of rural SSA women to ANC services [16]. Other reasons given for non-use of ANC services included ignorance and lack of money. This confirms similar observations from other rural African areas [20]. Interestingly, the Burkinabé Government had recently changed its policy towards free ANC service provision. However, our findings show that this policy is rather confusing as some parts of ANC procedures are not included. Consequently it was not yet fully understood by the population. However, this change in policy was considered promising during our interviews, and it was also reassuring that most of the ANC users were satisfied with the quality of ANC services. Knowledge and compliance with preventive interventions related to malaria and anaemia Malaria and anaemia were seen as important disease entities during pregnancy in our interviews. Moreover, most women interviewed were quite knowledgeable about effective malaria/anaemia prevention measures. However, compared to ANC users ANC non-users were significantly less knowledgeable about malaria/anaemia prevention measures. The interpretation is not straight forward because more knowledgeable women may be more likely to attend antenatal care or increased knowledge may be the result of health education in anatenatal care; most likely both effects contribute to the observed gap between ANC users and ANC non-users. Responsiveness of health services There is now a broad agreement on the need of new strategies for community participation on the implementation of effective malaria control activities, as well as for a better education of service providers of both the public and the private sector [17]. It was reassuring to find nearly all of the women with reported ANC use to have an ANC card in their house. Specific prescriptions were found on most cards, but in particular malaria prophylaxis prescriptions were often not complete. This explains why self-reported compliance with recommended prevention regimens was sub-optimal with regard to malaria. Such discrepancies have been observed already in a recent study in pregnant women of Nouna town [18]. However, self-reported compliance matched well with the reported oral instructions given by respective health workers. This points to the importance of correct oral information in rural areas with high prevalence of illiteracy [19]. The best model for effective and cost-effective malaria prevention during pregnancy in SSA still needs to be developed. Chloroquine has been the mainstay for malaria control in sub-Saharan Africa (SSA), but the emergence of chloroquine-resistant Plasmodium falciparum has put into question the efficacy of this well-known drug [21]. The first cases of in vitro and in vivo Chloroquine resistance in Burkina Faso were seen in 1983 and 1988, respectively, and reported clinical failure rates after use of Chloroquine for treatment of uncomplicated malaria in children were around 5% in the early 1990s and 10% during the most recently performed surveys [22]. Although this is considered still below the threshold of clinical failures considered to require a change of first-line treatment, it has recently been shown that chloroquine failed to prevent malaria in pregnant women of Burkina Faso [23]. Current alternatives include intermittent treatment with pyrimethamine-sulfadoxine and the use of ITNs. Sulphadoxine-pyrimethamine, given in 1–3 therapeutic dosages during the second and third trimester of pregnancy, has recently been demonstrated to be an effective and cost-effective schedule for malaria prevention [24]. Compared to chloroquine prophylaxis, this regimen has the advantage that it is given to women when they attend antenatal clinics, thus avoiding problems with compliance. ITNs are increasingly considered as an important tool in the prevention of malaria in young children and pregnant women, and the provision of ITN through ANC services has recently been proposed as a promising distribution channel [13,25]. The findings of this study confirm the cost barrier to the private purchase of bed nets and ITNs and thus support the call for major subsidies if a high ITN coverage is going to be achieved [12,13,25]. Competing interests None declared. Authors'Contributions CM, AJ and OM designed the study. FS and BK were responsible for the conduct of the study in Burkina Faso. CM analysed the data. All authors contributed to the interpretation of the data, helped write the paper, and read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank the staff of the CRSN and the population of the study villages for their support. The study was funded by the Deutsche Forschungsgemeinschaft (SFB 544, Control of Tropical Infectious Diseases). Caroline Miaffo was supported by a grant from the German Academic Exchange Service (DAAD). ==== Refs Steketee R Nahlen B Parise M Menendez C The burden of malaria in pregnancy in malaria-endemic areas Am J Trop Med Hyg 2001 64 28 35 11425175 Menendez C Malaria during Pregnancy: a priority area of malaria research and control Parasitology Today 1995 11 178 183 15275350 10.1016/0169-4758(95)80151-0 Garner P Gülmezoglu A Prevention versus treatment for malaria in pregnant women Cochrane Review The Cochrane Library 2001 Oxford: Update Software Shulman CE Marshall T Dorman EK Bulmer JN Cutts F Peshu N Marsh K Malaria in pregnancy: adverse effects on haemoglobin levels and birthweight in primigravidae Trop Med Int Health 2001 6 770 778 11679125 10.1046/j.1365-3156.2001.00786.x Bouvier P Breslow N Doumbo O Robert CF Picquet M Mauris A Dolo A Dembele HK Delley V Rougement A Seasonality, malaria, and impact of prophylaxis in a West African village II. Effect on birthweight Am J Trop Med Hyg 1997 56 384 389 9158045 WHO Implementation of the Global Malaria Control Strategy WHO Technical Report Series 839 1993 World Health Organisation Geneva Helitzer-Allen DL McFarland DA Wirima JJ Macheso AP Malaria chemoprophylaxis compliance in pregnant women: a cost-effectiveness analysis of alternative interventions Soc Sci Med 1993 36 403 407 8434265 10.1016/0277-9536(93)90402-P Steketee R Wirima JJ Campbell C Developing effective strategies for malaria prevention programs for pregnant African women Am J Trop Med Hyg 1996 55 95 100 8702046 Lengeler C Insecticide treated bednets and curtains for malaria control The Cochrane Library 1998 Oxford: Update Software Ter Kuile F Terlouw DJ Phillips-Howard PA Hawley WA Friedman JF Kariuki SK Shi YP Kolczak MS Lal AA Vulule JM Nahlen BL Reduction of malaria during pregnancy by permethrin-treated bed nets in an area of intense perennial malaria transmission in Western Kenya Am J Trop Med Hyg 2003 68 50 60 12749486 Müller O Becher H Baltussen A Ye Y Diallo D Konate M Gbangou A Kouyate B Garenne M Effect of zinc supplementation on malaria morbidity among West African children: A randomized double-blind placebo-controlled trial British Medical Journal 2001 322 1567 1572 11431296 Okrah J Traoré C Pale A Sommerfeld J Müller O Community factors associated with malaria prevention by mosquito nets: An exploratory study in rural Burkina Faso Trop Med Int Health 2002 7 240 248 11903986 10.1046/j.1365-3156.2002.00856.x Müller O Jahn A Expanding insecticide-treated mosquito net coverage in Africa: tradeoff between public and commercial strategies Trop Med Int Health 2003 8 853 856 14516294 10.1046/j.1365-3156.2003.01119.x Sankoh OA Ye Y Sauerborn R Müller O Becher H Clustering of childhood mortality in rural Burkina Faso International Journal of Epidemiology 2001 30 485 492 11416070 10.1093/ije/30.3.485 WHO World malaria situation in 1994 Weekly Epidemiol Rec 1997 72 269 292 Myer L Harrisson A Why do women seek antenatal care late? Perspectives from rural South Africa J Midwifery Womens Health 2003 48 268 272 12867911 10.1016/S1526-9523(02)00421-X Tanner M Vlassoff C Treatment-seeking behaviour for malaria: a typology based on endemicity and gender Soc Sci Med 1998 46 523 532 9460831 10.1016/S0277-9536(97)00195-0 Mietens A Anaemia and malaria in late pregnancy and the puerperium in Nouna District PhD thesis 2001 University of Heidelberg, Germany Yeneneh H Gyorkos TW Joseph L Pickering J Tedla S Antimalaria drug utilisation in Etiopia: a knowledge-attitudes-practice study Bulletin WHO 1993 71 762 772 Adamu YM Salihu HM Barriers to the use of antenatal and obstetric care services in rural Kano, Nigeria J Obstet Gynaecol 2002 22 600 603 12554244 10.1080/0144361021000020349 Trape JF The public health impact of chloroquine resistance in Africa Am J Trop Med Hyg 2001 64 12 17 11425173 Guigemdé TR Aoba A Ouedrago JB Lamizana L Ten Year surveillance of drug resistant malaria in Burkina Faso (1982–1991) Am J Trop Med Hyg 1994 50 699 704 8024062 Sirima SB Sawadogo R Moran AC Konate A Diarra A Yameogo M Parise ME Newman RD Failure of a chloroquine chemoprophylaxis program to adequately prevent malaria during pregnancy in Koupéla District, Burkina Faso Clin Infect Dis 2003 36 1374 1382 12766831 10.1086/375077 Newman RD Parise ME Slutsker L Nahlen B Steketee RW Safety, efficacy and determinants of effectiveness of antimalarial drugs during pregnancy: implications for prevention programmes in plasmodium falcipaum-endemic sub-saharan Africa Trop Med Int Health 2003 8 488 506 12791054 10.1046/j.1365-3156.2003.01066.x D'Alessandro U Coosemans M Is it feasible to give insecticide-treated bednets free to pregnant women? 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==== Front BMC PediatrBMC Pediatrics1471-2431BioMed Central London 1471-2431-4-161533933810.1186/1471-2431-4-16DebateSyndromes with congenital brittle bones Plotkin Horacio [email protected] Inherited Metabolic Diseases Section, Department of Pediatrics, University of Nebraska Medical Center, And Children's Hospital, Omaha, Nebraska, USA2004 31 8 2004 4 16 16 18 5 2004 31 8 2004 Copyright © 2004 Plotkin; licensee BioMed Central Ltd.2004Plotkin; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background There is no clear definition of osteogenesis imperfecta (OI). The most widely used classification of OI divides the disease in four types, although it has been suggested that there may be at least 12 forms of OI. These forms have been named with numbers, eponyms or descriptive names. Some of these syndromes can actually be considered congenital forms of brittle bones resembling OI (SROI). Discussion A review of different syndromes with congenital brittle bones published in the literature is presented. Syndromes are classified in "OI" (those secondary to mutations in the type I pro-collagen genes), and "syndromes resembling OI" (those secondary to mutations other that the type I pro-collagen genes, identified or not). A definition for OI is proposed as a syndrome of congenital brittle bones secondary to mutations in the genes codifying for pro-collagen genes (COL1A1 and COL1A2). Summary A debate about the definition of OI and a possible clinical and prognostic classification are warranted. classificationosteogenesis imperfectabrittle bonescongenital ==== Body Background Besides brittle bones, all other clinical characteristics of osteogenesis imperfecta (OI) are variable, and even different members of the same family may present with a dissimilar degree of severity [1]. Severity appears to follow a continuum in the OI population, and it is therefore very difficult to establish a clinical prognostic classification in definite categories. This disease has received different names through history, including osteopsathyrosis, Vrolik's disease, fragilitas ossium, mollities ossium, Lobstein's disease, and Van der Hoeve syndrome [2]. In a first attempt to classify OI, in 1906 Looser divided the disease in two forms [3]: "congenita" (Vrolik) and "tarda" (Lobstein), depending on the severity of the presentation. In OI congenita, multiple fractures may occur in-utero, whereas in OI tarda, fractures happen at the time of birth or later. OI tarda has also been sub-divided in "gravis" and "levis" [3]. This classification is no longer tenable because it failed to encompass the obvious clinical variability apparent in this disorder. There has also been an attempt to classify OI according to radiological characteristics [4]. Some of the features suggested by the authors of this classification are not present until age 5 or 10 years, and children of early ages can not be classified using this scheme. There has also been suggested that OI could be classified OI according to severity [5,6]. The most commonly used classification, initially proposed by Sillence in 1979, divides the patients in four types (I-IV) [7,8]. In fact, it is now widely recognized that there may be more types of OI [9-11]. Some of the forms of congenital brittle bones have been considered OI and have been added as numbers V, VI and VII [8,12-14], while others have been designated with eponyms (Cole-Carpenter syndrome [15], Bruck syndrome [16]), and others designated with clinical features (OI with denser areas in bones [17], OI with optic atrophy, retinopathy, and severe psychomotor retardation [18], OI with microcephaly, and cataracts [19], osteoporosis-pseudoglioma syndrome). The numeric classification (I-VII) is somewhat confusing, as the characteristics of each type overlap. Furthermore, there is no consensus about basic characteristics of the different types. For example, it is not clear if type I OI includes individuals with short stature, or if bone deformity rules out the diagnosis of Type I OI. Also, it is unclear if an individual with normal height can have type IV OI. Type II OI is defined as lethal, but there are cases of children with clinical characteristics of type II OI who have survived several years. Furthermore, the current classification does not allow for prognostic predictions, as individuals with type I OI may have numerous fractures and chronic pain in the course of their lives. This is part of the lack of a precise definition of OI. Thus, despite the fact that OI has been known for more than 200 years, there is no consensus about the definition for the disease. For some, it includes only those forms of congenital brittle bones secondary to mutations in the genes codifying for type I pro-collagen (COL1A1 and COL1A2). For others, it is a group of conditions with the common feature of congenital brittle bones. The example of the osteogenesis imperfecta-pseudoglioma syndrome illustrates this concept very clearly. This syndrome has been re-named as "osteoporosis-pseudoglioma syndrome" once the causal mutation has been identified to be in the LRP5 gene, elsewhere than the type I pro-collagen genes [20,21]. Following this example, I propose to define osteogenesis imperfecta as syndromes resulting from mutations in either COL1A1 or COL1A2 genes, and to group all other syndromes with congenital brittle bones as "syndromes resembling OI" (SROI), pending the identification of their causal mutation. Here, a review of the different forms of syndromes with congenital brittle bones is presented (Table 1). A working-group of international experts (like the Villefrance criteria in Ehlers-Danlos Syndrome) to clarify the issue of definition of congenital brittle bones syndromes and their classification is warranted. Table 1 syndromes with congenital brittle bones Osteogenesis Imperfecta  Mild OI with normal stature  Moderate OI with short stature  Severe OI  Lethal OI  Congenital brittle bones with dense areas in bones Syndromes resembling OI (SROI)  Congenital brittle bones with craniosynostosis and ocular proptosis  Congenital brittle bones with congenital joint contractures  Osteoporosis-pseudoglioma syndrome  Congenital brittle bones with optic atrophy, retinopathy and severe psychomotor retardation  Congenital brittle bones with microcephaly and cataracts  Congenital brittle bones with redundant callus  Congenital brittle bones with mineralization defect  Congenital brittle bones with rhizomelia Discussion Here, syndromes with congenital brittle bones are divided in osteogenesis imperfecta (those secondary to mutations in the type I pro-collagen genes) and syndromes resembling OI (those secondary to mutations other than the type I pro-collagen genes, identified or not) Osteogenesis imperfecta (types are ordered based on severity from mild to severe) Type I OI The most common mutation causing OI type I causes a reduction in the production of otherwise normal type I collagen secondary to the effect of a null allele mutation. Patients often have normal stature, and a slightly low stature does not preclude the diagnosis of type I OI. "Type I OI" is not a synonymous of "mild OI". Individuals may have few or no fractures, mostly during the first years of life or even at birth [22], or numerous fractures throughout their lives. They may have triangular face. They are fully ambulatory, and do not have bowing of the long bones, although vertebral fractures may be present. Most have blue sclera, but it can be white, or blue color may fade as the individual grows older. This condition is transmitted as an autosomal dominant trait. Despite the absence of fractures, bone density can be very low, with no relation with clinical severity, underscoring the relative lack of significance of bone density measurements assessing severity in patients with OI. In many instances bone density is normal during the first months of life, and individuals progressively fail to increase bone mineral density with age. In some cases the diagnosis is an incidental finding after a fracture [23]. Dentinogenesis imperfecta can be present even in very mild cases. Early hypoacusia [24,25] and cardio-vascular problems, particularly aortic valvular disease [22] can be present in these subjects. Type IV OI (Moderate OI with short stature) These individuals typically have short stature, bowing of long bones, and vertebral fractures. Scoliosis and joint laxity may be present. Patients with this form of OI are generally able to ambulate, but they may require aids for walking. Based in the presence of DI, moderate OI has been sub-divided in two forms, "a" and "b" [26]. These patients have white sclera. Precise diagnosis of this type of OI is often difficult, as the clinical characteristics are not clear in the literature, and different centers base the diagnosis on different criteria. Type III OI (Severe OI) These patients have triangular face, product of an enlarged head and under-development of the facial bones. They also have chest deformities, markedly short stature, and severe deformities of the long bones, vertebral fractures, and scoliosis. They are frequently wheelchair-bound, although some are able to walk with aids. Prenatal diagnosis is hard but sometimes possible using ultrasonography [27]. Long bones are severely bowed, and altered structure of the growth plates lead to a particular structural alteration of the metaphyses and epiphyses described a "popcorn appearance". Severe cases may have respiratory complications that can compromise survival. Type II OI In this form of OI, most newborns do not survive the perinatal period. Causes of death are malformations or hemorrhages of the central nervous system [28], extreme fragility of the ribs, or pulmonary hypoplasia [29]. The infants present with multiple intra-uterine fractures, including skull, long bones and vertebrae, beaded ribs, and severe deformity of the long bones [30]. Prenatal differential diagnosis between severe and lethal OI is usually not possible. Extremely severe cases can be dismembered during delivery [31]. The vast majority of cases are autosomal dominant new mutations [32-34]. It has been suggested that there may be different clinical forms of lethal OI [35]. Despite severity, a few patients have survived for several years. Congenital brittle bones with dense areas in bones Described in one infant [17] who died shortly after birth and presented with an OI phenotype that differed from the usual lethal form. The skeleton had regions of increased bone density, and this girl had dysmorphic facial features, including loss of mandibular angle, low set ears, soft skull, and large anterior and posterior fontanelles. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The metaphysis of the long bones were dense in x-rays. The patient died after a few hours and histopathological studies identified extramedullary hematopoiesis in the liver, little lamellar bone formation, decreased number of osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. Genetic testing showed that the child was heterozygous for a COL1A14321G → T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proa1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period [17]. Because of this mutation, this form of congenital brittle bones should be included in the OI group. Syndromes resembling OI (SROI) Congenital brittle bones with craniosynostosis and ocular proptosis (Cole-Carpenter syndrome) Two boys [15] and a girl [36] with this particular form of SROI have been described in the literature. Both boys were normal at birth, but after several months, they developed multiple metaphyseal fractures, associated with low bone density in the entire skeleton and craniosynostosis, hydrocephalus, ocular proptosis, and facial dysmorphism. One of the patients had also hypercalciuria. Neurological development is normal in this form of SROI. Both patients where wheelchair-bound at adult age, with very short stature, severe bone involvement and normal intellectual and neurological development [10]. No mutation has been identified as causing this syndrome. Congenital brittle bones with congenital joint contractures (Bruck syndrome) First described by Bruck et al in 1897 in an adult patient [16], in this form of SROI patients are born with brittle bones, leading to multiple fractures and joint contractures and pterygia (arthrogryposis multiplex congenita) due to dislocation of the radial head [37,38]. Wormian bones are present, and inheritance appears to be recessive [39,40]. In three patients that underwent pro-collagen mutation testing, it was not possible to demonstrate any mutations in the COL1A1 and COL1A2 genes [38]. The basic defect was mapped to locus 17p12 (18 cM interval), where a bone telopeptidyl hydroxylase is located [41]. The mutation leads to underhydroxilated lysine residues within the telopeptides of collagen type I, and therefore to aberrant crosslinking in bone, but not in cartilage or ligaments. Osteoporosis pseudoglioma syndrome [20,21] This form of SROI was first described in three families in 1972 [42]. Other report described a South African family of Indian origin with the condition [43]. Inheritance is autosomal recessive. Individuals with Osteoporosis-pseudoglioma syndrome have mild to moderate OI with blindness due to hyperplasia of the vitreous, corneal opacity and secondary glaucoma. The ocular pathology may be secondary to failed regression of the primary vitreal vasculature during fetal growth [44]. The genetic defect has been mapped to chromosome region 11q12-13 [45]. The defect is specifically in the LRP5 gene that encodes for the low-density lipoprotein receptor-related protein 5 [44]. Treatment with pamidronate has shown promising results in this group of patients [46]. Other forms of SROI with ocular involvement Two other forms of SROI with ocular involvement have been described: one variant with optic atrophy, retinopathy, and severe psychomotor retardation [18], another with microcephaly and cataracts [19]. Congenital brittle bones with redundant callus formation Originally published as "type V OI" [12]. Patients with this syndrome develop hyperplastic calluses in long bones after a fracture or intramedullary rodding surgery [22]. These patients present with hard, painful and warm swellings over long bones which initially may suggest inflammation or osteosarcoma. After a rapid growth period, the size and shape of the callus may remain stable for many years [47]. Microscopically there is increased production of abnormal extracellular matrix, that is poorly organized and incompletely mineralized in the callus area [48]. Histological studies in bone outside the callus area showed that the bone lamellae are arranged in a mesh-like fashion, as opposed to a parallel arrangement in patients with OI [12]. A series of case reports of hyperplastic callus formation in patients with clinical characteristics compatible with OI can be found in the literature [3,47,49-54]. These large calluses may also be present in flat bones [55]. These patients may also have calcification of the interosseous membrane between the radius and ulna, determining a clinical sign, as affected individuals are unable to pronate and supinate the forearm. The radial head may be dislocated. Patients with this syndrome have white sclera and no DI. Mutations in the pro-collagen genes could not be identified so far. Inheritance appears to be autosomal dominant, with variable penetrance. Congenital brittle bones with mineralization defect Undistinguishable from moderate to severe OI on a clinical basis, this rare form of SROI [13], has been classified as "Type VI OI". It can only be diagnosed by bone biopsy, where a mineralization defect affecting the bone matrix and sparing growth cartilage is evident. These patients have no DI and no wormian bones. Despite the histological mineralization defect, there are no radiological signs of growth plate involvement. The pattern of inheritance is not clear, but the case of two siblings from healthy consanguineous parents has been described, suggesting gonadal mosaicism or a somatic recessive trait [13]. No mutations of COL1A1 and COL1A2 genes have been found in these patients, and collagen structure appears to be normal. This form of SROI shares several characteristics with fibrogenesis imperfecta ossium [56,57]. Congenital brittle bones with rhizomelia A particular form of SROI with short humerus and recessive inheritance was described in a First Nations community of Quebec, and published as "type VII OI" [14]. The individuals affected have short humeri and femora. The phenotype is clinically moderate to severe. Fractures may be present at birth, and the condition progresses to early lower limb deformities, coxa vara and osteopenia. The bone morphology in congenital brittle bones with rhizomelia is not different than that of mild OI by histomorphometry. The genetic defect has been mapped to the short arm of chromosome 3 by linkage studies [58], where there are no genes that codify for type I pro collagen. Summary A definition for osteogenesis imperfecta is proposed based on the presence of type I pro-collagen mutations. Inclusion of related syndromes with unidentified mutations should be considered as SROI until further defined. Different forms of syndromes with congenital brittle bones are reviewed. A debate about the definition of OI and a possible clinical and prognostic classification are warranted. List of abbreviations OI Osteogenesis Imperfecta SROI syndromes resembling osteogenesis imperfecta DI Dentinogenesis imperfecta Competing interests None declared. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Smith R Osteogenesis imperfecta: from phenotype to genotype and back again Int J Exp Pathol 1994 75 233 241 7947227 Smith R Francis MJO Houghton GR The brittle bone syndrome 1983 London: Butterworths King JD Boblechko WP Osteogenesis imperfecta: an orthopaedic description and surgical review The Journal of Bone and Joint Surgery 1971 53B 72 89 Hanscom DA Winter RB Lutter L Lonstein JE Bloom BA Bradford DS Aldred MJ Osteogenesis imperfecta. Radiographic classification, natural history, and treatment of spinal deformities J Bone Joint Surg [Am] 1992 74 598 616 1583055 Albright JA Grunt JA Studies of patients with osteogenesis imperfecta J Bone Joint Surg [Am] 1971 53 1415 25 5114704 Bauze RJ Smith R Francis MJ A new look at osteogenesis imperfecta. A clinical, radiological and biochemical study of forty-two patients J Bone Joint Surg [Br] 1975 57 2 12 1117018 Sillence DO Senn A Danks DM Genetic heterogeneity in osteogenesis imperfecta J Med Genet 1979 16 101 16 458828 Sillence D Osteogenesis imperfecta: an expanding panorama of variants Clin Orthop 1981 11 25 7285446 Sillence D Osteogenesis Imperfecta 2000 Bone 2000 27 4s 10.1016/S8756-3282(00)89007-9 Plotkin H Primorac D Rowe D Glorieux F, Pettifor J, Juppner J Osteogenesis Imperfecta Pediatric Bone – Biology and Disease 2003 San Diego, CA: Elsevier Science 443 471 Plotkin H Lutz R Deng HW, Liu Y Osteoporosis in pediatrics Osteoporosis: Basic and Clinical Aspects 2004 Omaha: World Scientific Publishing Glorieux FH Rauch F Plotkin H Wart L Travers R Roughley P Lalic L Glorieux DF Fassier F Bishop NJ Type V osteogenesis imperfecta: a new form of brittle bone disease J Bone Miner Res 2000 15 1650 1658 10976985 Glorieux FH Wart L Rauch F Lalic L Roughley P Travers R Osteogenesis Imperfecta Type VI: a form of brittle bone disease with a mineralization defect Journal of Bone and Mineral Research 2002 17 30 38 11771667 Ward L Rauch F Travers R Chabot G Azouz E Lalic L Roughley PJ Glorieux FH Osteogenesis imperfecta type VII: an autosomal recessive form of brittle bone disease Bone 2002 31 12 18 12110406 10.1016/S8756-3282(02)00790-1 Cole DE Carpenter TO Bone fragility, craniosynostosis, ocular proptosis, hydrocephalus, and distinctive facial features: a newly recognized type of osteogenesis imperfecta J Pediatr 1987 110 76 80 3794889 Blacksin MF Pletcher BA David M Osteogenesis imperfecta with joint contractures: bruck syndrome Pediatr Radiol 1998 28 117 9 9472060 10.1007/s002470050309 Pace JM Chitayat D Atkinson M Wilcox WR Schwarze U Byers PH A single amino acid substitution (D1441Y) in the carboxyl-terminal propeptide of the proalpha1(I) chain of type I collagen results in a lethal variant of osteogenesis imperfecta with features of dense bone diseases J Med Genet 2002 39 23 9 11826020 10.1136/jmg.39.1.23 al Gazali LI Sabrinathan K Nair KG A syndrome of osteogenesis imperfecta, optic atrophy, retinopathy and severe developmental delay in two sibs of consanguineous parents Clin Dysmorphol 1994 3 55 62 7515752 Buyse M Bull MJ A syndrome of osteogenesis imperfecta, microcephaly, and cataracts Birth Defects Orig Artic Ser 1978 14 95 98 728577 Beighton P Osteoporosis-pseudoglioma syndrome [letter] Clin Genet 1986 29 263 3698336 Capoen J De Paepe A Lauwers H The osteoporosis pseudoglioma syndrome J Belge Radiol 1993 76 224 5 8294375 Smith R Osteogenesis imperfecta Clinics in Rheumatic Diseases 1986 12 655 89 3078778 Match RM Corrylos EV Bilateral avulsion fracture of the triceps tendon insertion from skiing with osteogenesis imperfecta tarda. A case report American Journal of Sports Medicine 1983 11 99 102 6846689 Shapiro JR Pikus A Weiss G Rowe DW Hearing and middle ear function in osteogenesis imperfecta JAMA 1982 247 2120 2126 7062527 10.1001/jama.247.15.2120 Pedersen U Hearing loss in patients with osteogenesis imperfecta. A clinical and audiological study of 201 patients Scand Audiol 1984 13 67 74 6463554 Levin LS Salinas CF Jorgenson RJ Classification of osteogenesis imperfecta by dental characteristics [letter] Lancet 1978 1 332 3 75372 10.1016/S0140-6736(78)90108-3 Robinson LP Worthen NJ Lachman RS Adomian GE Rimoin DL Prenatal diagnosis of osteogenesis imperfecta type III Prenatal Diagnosis 1987 7 7 15 3547385 Pauli RM Gilbert EF Upper cervical cord compression as cause of death in osteogenesis imperfecta type II J Pediatr 1986 108 579 581 3958833 Shapiro JR Burn VE Chipman SD Jacobs JB Schloo B Reid L Larsen N Louis F Pulmonary hypoplasia and osteogenesis imperfecta type II with defective synthesis of alpha I(1) procollagen Bone 1989 10 165 171 2803853 10.1016/8756-3282(89)90049-5 Andrews M Amparo EG In utero clue to congenital lethal osteogenesis imperfecta [letter] AJR Am J Roentgenol 1993 160 212 8416637 Heller RH Winn KJ Heller RM The prenatal diagnosis of osteogenesis imperfecta congenita Am J Obstet Gynecol 1975 121 572 3 1146889 Prockop DJ Mutations in collagen genes. Consequences for rare and common diseases J Clin Invest 1985 75 783 7 3884665 Byers PH Tsipouras P Bonadio JF Starman BJ Schwartz RC Perinatal lethal osteogenesis imperfecta (OI type II): a biochemically heterogeneous disorder usually due to new mutations in the genes for type I collagen American Journal of Human Genetics 1988 42 237 248 3341380 Cole WG Dalgleish R Perinatal lethal osteogenesis imperfecta J Med Genet 1995 32 284 289 7643358 Sillence DO Barlow KK Garber AP Hall JG Rimoin DL Osteogenesis imperfecta type II delineation of the phenotype with reference to genetic heterogeneity Am J Med Genet 1984 17 407 23 6702894 Amor DJ Savarirayan R Schneider AS Bankier A New case of Cole-Carpenter syndrome American Journal of Medical Genetics 2000 92 273 277 10842295 10.1002/(SICI)1096-8628(20000605)92:4<273::AID-AJMG10>3.0.CO;2-T McPherson E Clemens M Bruck syndrome (osteogenesis imperfecta with congenital joint contractures): review and report on the first North American case Am J Med Genet 1997 70 28 31 9129737 10.1002/(SICI)1096-8628(19970502)70:1<28::AID-AJMG6>3.3.CO;2-N Leroy JG Nuytinck L De Paepe A De Rammelaere M Gillerot Y Verloes A Loeys B De Groote W Bruck syndrome: neonatal presentation and natural course in three patients Pediatric Radiology 1998 28 781 9 9799301 10.1007/s002470050465 Viljoen D Versfeld G Beighton P Osteogenesis imperfecta with congenital joint contractures (Bruck syndrome) Clin Genet 1989 36 122 126 2766569 Brady AF Patton MA Osteogenesis imperfecta with arthrogryposis multiplex congenita (Bruck syndrome)–evidence for possible autosomal recessive inheritance Clin Dysmorphol 1997 6 329 36 9354841 Bank RA Robins SP Wijmenga C Breslau-Siderius LJ Bardoel AFJ Van der Sluijs HA Pruijs HEH TeKoppele JM Defective collagen crosslinking in bone, but not in ligament or cartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase on chromosome 17 Proc Natl Acad Sci USA 1999 96 1054 1058 9927692 10.1073/pnas.96.3.1054 Bianchine JW Briard-Guillemot ML Maroteaux P Frezal J Harrison HE Generalized osteoporosis with bilateral pseudoglioma – an autosomal recessive disorder of connective tissue: report of three families – review of the literature Am J Hum Genet 1972 24 34A Beighton P Winship I Behari D The ocular form of osteogenesis imperfecta: a new autosomal recessive syndrome Clin Genet 1985 28 69 75 4028503 Gong Y Slee RB Fukai N Rawadi G Roman-Roman S Reginato AM Wang H Cundy T Glorieux FH Lev D LDL Receptor-related protein 5 (LRP5) affects bone accrural and eye development Cell 2001 107 513 523 11719191 10.1016/S0092-8674(01)00571-2 Gong Y Vikkula M Boon L Beighton P Ramesar R Peltonen L Somer H Hirose T Dallapiccola B De Paepe A Osteoporosis-pseudoglioma syndrome, a disorder affecting skeletal strength and vision, is assigned to chromosome region 11q12-13 American Journal of Human Genetics 1996 59 146 151 8659519 Zacharin M Cundy T Osteoporosis pseudoglioma syndrome: Treatment of spinal osteoporosis with intravenous bisphosphonates J Pediatr 2000 137 410 415 10969269 10.1067/mpd.2000.107838 Banta JV Schreiber RR Kulik WJ Hyperplastic callus formation in osteogenesis imperfecta simulating osteosarcoma J Bone Joint Surg [Am] 1971 53 115 22 5276046 Morike M Windsheimer E Brenner R Nerlich A Bushart G Teller W Vetter U Effects of transforming growth factor beta on cells derived from bone and callus of patients with osteogenesis imperfecta J Orthop Res 1993 11 564 72 8340828 Roberts JB Bilateral hyperplastic callus formation in osteogenesis imperfecta J Bone Joint Surg [Am] 1976 58 1164 6 1002764 Burke TE Crerand SJ Dowling F Hypertrophic callus formation leading to high-output cardiac failure in a patient with osteogenesis imperfecta J Pediatr Orthop 1988 8 605 8 3170743 Burchardt AJ Wagner AA Basse P Hyperplastic callus formation in osteogenesis imperfecta. A case report Acta Radiol 1994 35 426 8 8086246 Azouz EM Fassier F Hyperplastic callus formation in OI [letter] Skeletal Radiol 1997 26 744 5 9453112 10.1007/s002560050324 Kutsumi K Nojima T Yamashiro K Hatae Y Isu K Ubayama Y Yamawaki S Hyperplastic callus formation in both femurs in osteogenesis imperfecta Skeletal Radiol 1996 25 384 7 8738006 10.1007/s002560050100 Nakamura K Kurokawa T Nagano A Umeyama T Familial occurrence of hyperplastic callus in osteogenesis imperfecta Arch Orthop Trauma Surg 1997 116 500 3 9352048 10.1007/s004020050172 Dobrocky I Seidl G Grill F MRI and CT features of hyperplastic callus in osteogenesis imperfecta tarda European Radiology 1999 9 665 668 10354881 10.1007/s003300050729 Baker SL Dent CE Friedman M Watson L Fibrogenesis imperfecta ossium J Bone Joint Surg Br 1966 48 804 825 5954373 Lang R Vignery AM Jensen PS Fibrogenesim Imperfecta Ossium with early onset: observations after 20 years of illness Bone 1986 7 237 246 3490268 10.1016/8756-3282(86)90202-4 Labuda M Morissette J Wart LM Rauch F Lalic L Roughley PJ Glorieux FH Osteogenesis imperfecta type VII maps to the short arm of chromosome 3 Bone 2002 31 19 25 12110407 10.1016/S8756-3282(02)00808-6

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==== Front Biomagn Res TechnolBiomagnetic Research and Technology1477-044XBioMed Central London 1477-044X-2-51532914910.1186/1477-044X-2-5ResearchPerformance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed Yavuz Handan [email protected] Ridvan [email protected]ç Müge [email protected] Necmi [email protected] Adil [email protected] Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara, Turkey2 Department of Chemistry, Anadolu University, Ankara, Turkey3 Faculty of Medicine, Urology Department, Hacettepe University, Ankara, Turkey2004 26 8 2004 2 5 5 22 7 2004 26 8 2004 Copyright © 2004 Yavuz et al; licensee BioMed Central Ltd.2004Yavuz et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. Methods Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80–120 μm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 μmol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. Results Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. Conclusions Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium. ==== Body Background About 8% of the Earth's crust is comprised of aluminium. This element is the most abundant metal naturally present in air, soil and water. Consequently, environmental exposure to aluminium is potentially possible. Its ingestion is unavoidable since aluminium compounds are added not only to most water supplies but also to many processed foods and medicines. Aluminium is a known neurotoxicant. It enters the brain, where it contributes to some neuro-degenerative diseases including dialysis encephalopathy, osteomalacia, osteodystrophy, in particular those related to dialysis treatment of uremic subjects [1]. Only a small amount of Al(III) ions in dialysis solutions may cause these disorders. Aluminium may contribute to Alzheimer's disease [2]. Aluminium is also able to give rise to toxicity in the bones and hematopoietic system in humans [3]. Positively charged aqua and hydroxy-monomeric forms have been found to be the most toxic species of aluminium to living organisms in the terrestrial and aquatic environments [4]. Generally, aluminium sulphate is used as a coagulant in the treatment of water to help the removal of suspended matter and highly coloured humic substances [5], thus reducing the dose of chlorine later required to ensure satisfactory microbiological quality. Hence, potable water often contains high aluminium levels of natural origin and/or from the water purification process [6]. The selective removal of aluminium ions have been extensively investigated by applying several techniques [7-9]. Among them, the use of specific polymeric adsorbents has been considered as one of the most promising techniques [10,11]. Specific adsorbents consist of a ligand (e.g., reactive textile dye, ion-exchange functional groups or chelating agents) which interacts with the metal ions specifically, and a carrier solid matrix. There have been several separation approaches performed under magnetic field [12]. The most well known technique is the magnetically stabilized fluidized bed. Magnetically stabilized fluidized bed exhibits combination of the best characteristics of both packed and fluidized bed. These include the efficient fluid-solid mass transfer properties, elimination of particle mixing, low pressure drop, high feed-stream solid tolerances, good fluid-solid contact, elimination of clogging and continuous countercurrent operation [13]. Especially, when dealing with highly viscous mediums contact with the magnetic adsorbent in a magnetically stabilized fluidized bed is desirable because of high convective transport rates. Recently, there has been increased interest in the use of magnetic adsorbents in biomolecule coupling and nucleic acid purification [14]. Magnetic adsorbents can be produced using inorganic materials or polymers. High mechanical resistance, insolubility and excellent shelf life make inorganic materials ideal as adsorbent. The main disadvantage of inorganic supports is their limited functional groups for ligand coupling. Magnetic adsorbents can be porous or non-porous [15]. They are more commonly manufactured from polymers since they have a variety of surface functional groups which can be tailored to use in different applications [16-22]. In the present study, we attempted to use alizarin yellow-attached magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads as specific adsorbent for aluminium removal from aqueous solutions in a magnetically stabilized fluidized bed. Al(III) adsorption on the alizarin yellow-affinity beads from aqueous solutions containing different amounts of Al(III) ions and at different pHs is reported here. Finally, reuse of the dye-affinity beads is also discussed. Materials and methods Materials 2-hydroxyethyl methacrylate (HEMA), was purchased from Sigma (St. Louis, MO, USA), and was purified by vacuum distillation under a nitrogen atmosphere. The comonomer, ethylene dimethacrylate (EDMA, Merck, Darmstadt, Germany) was used as the crosslinking agent. Magnetite particles (Fe3O4, diameter < 1 μm) were obtained from Aldrich (USA). Alizarin yellow (3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt mono-hydrate) was purchased from BDH (Poole, UK). All other chemicals were obtained from Merck as analytical grade. All water used in the adsorption experiments was purified using a Barnstead (Dubuque, IA) ROpure LP® reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure® organic/colloid removal and ion exchange packed bed system. Preparation of magnetic PHEMA beads Details of the preparation and characterization of the mPHEMA beads were reported elsewhere [23]. The mPHEMA beads were prepared by suspension polymerization. A typical suspension copolymerization procedure of mPHEMA beads was performed as below: The dispersion medium was prepared by dissolving 200 mg of poly(vinyl alcohol) (PVA; molecular weight: 50.000) within 50 ml of distilled water. The desired amount of 2,2'-azobisisobutyronitrile (AIBN) (0.06 g) was dissolved within the monomer phase 12.0/4.0/8.0 ml (EDMA/HEMA/toluene) with 1.0 g magnetite particles. This solution was then transferred into the dispersion medium placed in a magnetically stirred (at a constant stirring rate of 600 rpm) glass polymerization reactor (100 ml) which was in a thermostatic water bath. The reactor was flushed by bubbling nitrogen and then was sealed. The reactor temperature was kept at 65°C for 4 h. The temperature was then raised to 90°C and kept constant by a thermostated water bath during the polymerization time (2 h). After polymerization, the mPHEMA beads were separated from the polymerization medium. The residuals (e.g., unconverted monomer, initiator and other ingredients) were removed by a cleaning procedure. Briefly, beads were transferred into a reservoir, and washing solutions (i.e., a dilute HCI solution, and a water-ethanol mixture) were recirculated through the system which includes also an activated carbon column, to be assured that the magnetic beads were clean. Purity of the magnetic beads was followed by observing the change of optical densities of the samples (λ: 280 nm) taken from the liquid phase in the recirculation system, and also from the DSC thermograms of the magnetic beads obtained by using a differential scanning microcalorimeter (Mettler, Switzerland). Optical density of uncleaned magnetic beads was 2.63, but after the cleaning operation this value was reduced to zero. In addition, when the thermogram of uncleaned beads was recorded, it had a peak around 60°C. This peak might originate from AIBN, but after application of the cleaning procedure, no peak between 30–100°C was observed on the thermogram. The dry density of the magnetic beads was measured with pycnometer by dispersing the dry beads in ethanol. Alizarin yellow attachment Preparation and characterization of the alizarin yellow-attached mPHEMA beads were reported in our previous paper in detail [24]. In order to prepare the alizarin yellow-attached magnetic beads following procedure was applied. 5.0 g of dry magnetic beads was weighed and transferred into the SOCl2 (Carlo Erba, Italy) (10 ml). This reaction medium was boiled in rotary evaporator for 6 h. Then, 2.5 g alizarin yellow was dissolved in absolute ethanol (30 ml). Alizarin yellow-attachment process was performed in ethanol solution for 24 h. At the end of this reaction period, the alizarin yellow-attached beads were removed by filtration and washed with ethanol, water and tetrahydrofuran several times until all the unbound dye molecules were removed. The dye attached beads were stored at 4°C with 0.02% sodium azide to prevent microbial growth. The leakage of the alizarin yellow from the dye-attached beads was investigated within the media at the selected pH in the range of 2.0–7.0. These media were the same which were used in the Al(III) adsorption experiments. The medium with the dye attached magnetic beads was stirred for 24 h at room temperature. Then, magnetic beads were separated from the medium, and the alizarin yellow concentration was measured in the liquid phase by spectrophotometry at 500 nm. Magnetically stabilized fluidized bed procedure Al(III) adsorption studies were carried out in a magnetically stabilized fluidized bed. Beads suspended in pure water were degassed under reduced pressure (by using water suction pump) and magnetically stabilized into a column (10 cm × 0.9 cm inside diameter) equipped with a water jacket for temperature control. The vertically oriented magnetic field was produced by passing DC current through two modified Helmholtz coils (1.5 cm diameter × 2.5 cm thick) spaced 4 cm apart. At a current of 1.6 A (50 W), each coil produced a magnetic field of 40 Gauss. Equilibration of the column was performed by passing four column volumes of phosphate buffer (pH: 7.4) before injection of the Al(III) solution. In a typical adsorption system, 50 ml of the aqueous Al(III) solution was passed through the column containing magnetic beads, by a peristaltic pump for 2 h. After loading, the column was washed with deionized water to wash out Al(III) impurities. The concentrations of the Al(III) ions in the aqueous phases after the desired treatment periods were measured by using a graphite furnace atomic absorption spectrophotometer (AAS 5EA, Carl Zeiss Technology, Zeiss Analytical Systems, Germany). Deuterium background correction was used. Pyrolitic graphite coated tubes were used for AAS measurements. The instrument response was periodically checked with known Al(III) solution standards. The experiments were performed in replicates of three and the samples were analyzed in replicates of three as well. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error. In the first group of experiments, the flow rate of the aqueous solution (i.e., 50 ml of the solution with a Al(III) content of 50 mg/L) was changed between 0.5–3.0 mL/min. In the second group of experiments, Al(III) adsorption from aqueous solution was studied at different pH's (2.0–7.0). Adsorption isotherm was also obtained in the magnetically stabilized fluidized bed. Aqueous solutions containing different amount of Al(III) were used in these experiments. The changes in the Al(III) concentration with time was followed to obtain the adsorption curves. The amount of adsorbed Al(III) per dry magnetic beads was calculated by using the concentrations of the Al(III) in the initial solution and in the equilibrium. Desorption and repeated use In all cases adsorbed Al(III) ions were desorbed using 0.1 M HNO3 solution. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the magnetically stabilized fluidized bed containing dye-affinity magnetic beads for 1 h. The concentrations of the Al(III) ions in the desorption medium were measured by using a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the amount of Al(III) adsorbed on the magnetic beads and the final Al(III) concentration in the desorption medium. In order to test the reusability of the dye-affinity magnetic beads, Al(III) adsorption-desorption procedure was repeated ten times by using the same magnetically stabilized fluidized bed. Batch procedure Adsorption of Al(III) from aqueous solution was also investigated in batch experiments. Aqueous Al(III) solution (50 ml) was treated with the magnetic dye-affinity beads at room temperature, in the flasks agitated magnetically at an agitation speed of 600 rpm for 2 h. The suspension was brought to pH 5.0 by adding sodium hydroxide and nitric acid. The pH was maintained in a range of ± 0.1 units until equilibrium was attained. Polymer amount was kept constant at 100 mg per 50 ml. Al(III) determination was performed in water sample in an atomic absorption spectrophotometer coupled to a graphite furnace atomiser. Adsorption values (mg/g) were calculated as the difference in Al(III) ion concentration of the pre- and post adsorption solutions divided by the weight of dry magnetic affinity beads. Results and discussion Characteristics of mPHEMA beads mPHEMA beads (in the size range of 80–120 μm) carrying alizarin yellow were prepared as a specific affinity adsorbent for removal of Al(III) from the water which was used for preparation of dialysis solution. mPHEMA beads used in this study were prepared and characterized in our earlier study [24]. The main criteria of selection of PHEMA is due to its mechanical strength and chemical stability. With the goal of testing the mechanical stability of the magnetic beads, a sample of these magnetic beads was treated in a ball mill for 60 min. Negligible percentage of the sample was broken. The dry density of the magnetic beads was measured as 1.27 g/cm3. The magnetic beads are crosslinked hydrogels. They do not dissolve in aqueous media, but do swell, depending on the degree of cross-linking and on the hydrophilicity of the matrix. The equilibrium swelling ratio (the ratio of the volumes of the microbeads before and after swelling) of the beads used in this study is 34%. The simple incorporation of water weaken the secondary bonds within the hydrogels. This enlarges the distance between the polymer chains and causes the uptake of water. It should be mentioned that the water uptake properties of the mPHEMA beads did not change after Alizarin Yellow attachment. After the attachment of the dye (i.e., alizarin yellow) the size of the swollen beads did not change, but the colour became dark yellow, which is a clear indication of the incorporation of the dye molecules in the structure of the mPHEMA microbeads. As shown in our previous paper, the dye molecules were attached to the mPHEMA beads by covalent bonding via hydroxyl groups [24]. The mPHEMA beads containing 208 μmol alizarin yellow/g polymer, which was the maximum amount of dye-attachment that we have reached, were used in this study. Alizarin Yellow release from the mPHEMA beads was also monitored continuously. There were no dye release in any of the adsorption and desorption media, which assured that the cleaning procedure used for removal of physically adsorbed alizarin yellow molecules from the mPHEMA beads was satisfactory. Column performance The adsorption capacity at different flow-rates are given in Figure 1. The adsorption capacity decreased significantly from 17.2 mg/g to 6.9 mg/g polymer with the increase of the flow-rate from 0.5 ml/min to 3.0 ml/min. One of the explanation for such phenomenon would be a faster ligand-metal ion (i.e., alizarin yellow) dissociation rate compared to the association rate. Hence, the adsorbate (i.e., Al(III) ions) would pass through the magnetically stabilized column without adsorption at high flow-rate. Second explanation could be that the increased nonideal flow hydrodynamics of liquid phase and the solid phase for magnetically stabilized fluidized bed. These phenomena can be summarized by the increase of the axial dispersion coefficient in the axial dispersion model [25]. Figure 1 Effect of flow-rate on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Al(III) concentration: 50 mg/L; pH: 5.0; T: 25°C. Adsorption capacity Figure 2 shows the adsorption profile of Al(III) ions. The amount of Al(III) ions adsorbed per unit mass of the polymer (i.e. adsorption capacity) increased first with the initial concentration of Al(III) ions then reached a plateau value at about an initial Al(III) ions concentrations of 50 mg/L, which represents saturation of the active attachment sites (which are available for Al(III) ions) on the beads. The maximum adsorption capacity of Al(III) ions was of 647 μmol/g (17.5 mg/g). Unit mass of the mPHEMA beads carries 208 μmol alizarin yellow which was found by elemental analysis. From the mass-stoichiometry, it seems that one attached alizarin yellow molecule interacts with around three Al(III) ions. Since alizarin yellow has seven coordinating sites of a single sulphur and six oxygen atoms, it can form a ternary complex which is coordinated with water molecules at vacant coordination sites of metal-alizarin yellow complexes. Figure 2 Effects of Al(III) concentration on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; pH: 5.0; Adsorption time: 60 min; T: 25°C. It should be noted that the nonspecific adsorption (adsorption on plain mPHEMA beads) of Al(III) ions was relatively low (0.63 mg/g). mPHEMA beads do not contain ion exchange or chelating groups. Preferred coordination structure and preferred coordinating ligand atom may be utilized for this adsorption. Al(III) ions may interact with Oxygen atoms as the ligand. Diffusion of Al(III) ions into the swollen polymeric structure and retention in the pores may also contribute to this nonspecific Al(III) adsorption. Adsorption isotherms An adsorption isotherm is used to characterize the interactions of each molecule with the adsorbent. In this case it provides a relationship between the concentration of the Al(III) ions in the solution and the amount of Al(III) ions adsorbed on the solid phase when the two phases are at equilibrium. The Langmuir adsorption model assumes that the species are adsorbed at a fixed number of well-defined sites, each of which is capable of holding only one molecule. These sites are also assumed to be energetically equivalent, and distant from each other so that there are no interactions between molecules adsorbed on adjacent sites. Adsorption isotherms were used to evaluate adsorption properties. The Langmuir adsorption isotherm is expressed by Equation 1. The corresponding transformations of the equilibrium data for Al(III) gave rise to a linear plot, indicating that the Langmuir model could be applied in these systems and described by the equation: Q = Qmax. b . Ceq / (1 + bCeq)     (1) where Q is the adsorbed amount of Al(III) (mg/g), Ceq is the equilibrium Al(III) concentration (mg/mL), b is the Langmuir constant (mL/mg) and, Qmax is the maximum adsorption capacity (mg/g). This equation can be linearized so that Ceq/Q = 1/(Qmax. b) + Ceq/Qmax.     (2) The plot of Ceq versus Ceq/Q was employed to generate the intercept of 1/Qmax.b and the slope of 1/Qmax. The maximum adsorption capacity (Qmax) data for the adsorption of Al(III) was obtained from the experimental data. The correlation coefficient (R2) was 0.989. The Langmuir adsorption model can be applied in this affinity adsorbent system. Maximum adsorption capacities determined using Langmuir isotherms show that dynamic capacity (25.3 mg/g) was somewhat higher than the batch capacity (12.6 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Effect of pH Metal ion adsorption onto specific adsorbents is pH dependent. In the absence of complexing agents, the hydrolysis and precipitation of the metal ions are affected by the concentration and form of soluble metal species. The solubility of metal ions is governed by hydroxide or carbonate concentration. Hydrolysis of metal ions becomes significant at approximately pH 7.5–8.5. Therefore, in the present study, we changed the pH range between 2.0–7.0. The effect of pH on the Al(III) adsorption of this alizarin yellow-attached mPHEMA beads is also shown in Figure 3. The magnetic mPHEMA beads exhibited a low affinity in acidic condition (pH < 4.0), a somewhat higher affinity between pH 4.0 and 7.0. High adsorption capacities at around neutral pH values imply that Al(III) ions interact with dye molecules not only through the oxygen atoms by chelating, but also electrostatically through sulfonate groups, which are ionized at neutral pH. Figure 3 Effects of pH on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; T: 25°C. Competitive adsorption Competitive adsorption of the metal ions from tap water in Ankara and dialysis water (reverse osmosis) was also investigated. The water containing different amounts of each metal ion was treated with dye beads in MSFB. Table 1 and 2 show the adsorbed amounts for each metal ion. The adsorption capacity of the dye-attached mPHEMA beads for Cu(II) and Al(III) ions was higher than that for other ions. But it should also be noted that the extent of adsorption of each type of metal ion is strongly dependent upon their relative concentrations within the medium. Table 1 Aluminium removal from the tap water. Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) Al(III) 80.1 40.3 ± 0.1 98.9 Fe(III) 32.1 10.8 ± 0.2 54.3 Cu(II) 145.3 28.3 ± 0.2 35.1 Cd(II) 0.05 nd - Pb(II) 0.03 nd - Zn(II) 20.4 1.5 ± 0.1 28.6 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C, nd: not determined. Each experiment was repeated three times. Table 2 Aluminium removal from dialysis water. Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) Al(III) 18.96 9.46 ± 0.1 99.7 Fe(III) 0.05 - - Cu(II) 0.82 0.16 ± 0.01 40.0 Zn(II) 1.26 0.44 ± 0.01 69.8 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C. Each experiment was repeated three times. The World Health Organization (WHO) and the European Community (EC) guide values for Al(III) ions for tap water is 200 ng/ml [26,27]. Al(III) concentrations both in tap water and dialysis water are below this value. It should be noted that polymer treatment (i.e, adsorption) significantly decreases the metal content and these purified waters can be used safely especially for the preparation of dialysis solutions. Magnetic dye-affinity beads exhibits the following metal ion affinity sequence: Al(III) > Cu(II) > Fe(III) > Zn(II). Desorption and repeated use Desorption ratios were very high (up to 97.6%) with the eluant system and under conditions used. When HNO3 is used as a desorption agent, the coordination spheres of chelated Al(III) ions are disrupted and subsequently Al(III) ions are released from the solid surface into the desorption medium. Therefore, we conclude that HNO3 is a suitable desorption agent for the dye adsorbents, and allows their repeated use. In order to show the reusability of the dye-attached mPHEMA beads, adsorption-desorption cycle was repeated ten times by using the same sample of affinity adsorbent. As can be seen from Figure 4, adsorption capacities did not noticeable change during the repeated adsorption-desorption cycles. Figure 4 Repeated use of dye-attached mPHEMA beads. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. Comparison of magnetically stabilized fluidized bed and batch system As can be seen in Figure 5, maximum Al(III) adsorption from aqueous solution is 11.8 mg/g for batch system and 17.5 mg/g for MSFB system. These results indicated that the adsorption capacity obtained in MSFB system is considerably higher than obtained in batch sytstem. This means, in equilibrium binding experiments, maximum capacity was 38.8% lower as compared to the value obtained in MSFB. This result could be explained in two ways. (i) The dye ligand-Al(III) dissociation rate in the batch system is higher than the association rate in the MSFB system. (ii) Alizarin yellow ligand is found both on the surface and in the pores of the magnetic beads. In the presence of flow, the Al(III) solution is forced from the surface into the pores thus eliminating the surface diffusion. Figure 5 Comparison of MSFB and batch system. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. Conclusions The medical relevance of aluminium has stimulated the development of cost and time effective separation techniques including polymeric carriers. Magnetic adsorbents have several potential advantages over conventional adsorbents [28-32]. The magnetically stabilized columns require faster processing times and high flow-rates with a much lower operating pressure than a packed bed column. In this study, mPHEMA beads, in the size fraction of 80–120 μm, were produced by a dispersion polymerization of EGDMA and HEMA in the presence of magnetite particles. These novel magnetic beads were then successfully attached with reactive dye-ligand, namely alizarin yellow. The highest dye loading was 208 μmol/g. Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 4.0. Al(III) adsorption onto the mPHEMA beads was negligible (0.63 mg/g). Higher adsorption values (up to 17.5 mg/g) were observed using alizarin yellow attached mPHEMA beads for the adsorption of Al(III) ions from aqueous solutions. Al(III) ions could be repeatedly adsorbed and desorbed without significant losses in their adsorption capacities. ==== Refs Martin RB The chemistry of aluminum as related to biology and medicine Clin Chem 1986 32 1797 1806 3019589 Wills MR Savory J Aluminium poisoning: dialysis encephalopathy Lancet 1983 ii 29 34 6134894 10.1016/S0140-6736(83)90014-4 Martyn CN Barker DJP Osmond C Harris EC Edwardson JA Lacey RF Geographical relation between Alzheimer's disease and aluminium in drinking water Lancet 1989 i 59 62 2562879 Nicolini M Zatta PF Carain B Eds Aluminum in Chemistry, Biology and Medicine Cortina International, Verona 1991 Plessi M Monzani A Aluminium determination in bottled mineral waters by electro-thermal atomic absorption spectrometry J Food Compos Anal 1995 8 21 26 10.1006/jfca.1995.1004 Chang TMS Barre P Effect of desferrioxamine on removal of aluminium and iron by coated charcoal haemoperfusion and haemodialysis Lancet 1983 ii 1051 6138600 10.1016/S0140-6736(83)91039-5 Ackrill P Ralston AJ Day JP Hodge KC Successful removal of aluminium from patient with dialysis encephalopathy Lancet 1980 ii 692 693 6106803 10.1016/S0140-6736(80)92728-2 Altmann P Plowman Marsh F Cunningham J Aluminium chelation therapy in dialysis patients: evidence for inhibition of haemoglobin synthesis by low levels of aluminium Lancet ii 1012 1014 Gerhardsson L Oskarsson A Skerfving S Acid precipitation-effects on trace elements and human health Sci Total Environ 1994 153 237 245 7939624 10.1016/0048-9697(94)90203-8 Denizli A Salih B Kavakli C Piskin E Dye-incorporated poly(EGDMA-HEMA) micro-spheres as specific sorbents for aluminium removal J Chromatogr B 1997 698 89 96 10.1016/S0378-4347(97)00306-X Say R Denizli A Preparation of magnetic dye affinity adsorbent and its use in the removal of aluminium ions J Biomater Sci Polym Ed 2001 12 1059 1073 11853378 10.1163/15685620152691850 Xue B Sun Y Protein adsorption equilibria and kinetics to a poly(vinyl alcohol)-based magnetic affinity support J Chromatogr A 2001 921 109 119 11471795 10.1016/S0021-9673(01)00860-3 Burns MA Kvesitadze GI Graves DJ Dried calcium alginate/magnetite spheres: a new support for chromatographic separations and enzyme immobilization Biotechnol Bioeng 1985 27 137 145 Bilkova Z Slovakova M Lycka A Horak D Lenfeld J Turkova J Churacek J Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres J Chromatogr B 2002 770 25 34 O'Brien SM Thomas ORT Dunnill P Nonporous magnetic chelator supports for protein recovery by immobilised metal affinity adsorption J Biotechnol 1996 50 13 25 10.1016/0168-1656(96)01544-1 Rad AY Yavuz H Denizli A Bilirubin removal from human plasma with albumin immobilised magnetic poly(2-Hydroxyethyl methacrylate) beads Macromol Biosci 2003 3 471 476 10.1002/mabi.200350018 Martin C Cuellar J Synthesis of a novel magnetic resin and the study of equilibrium in cation exchange with amino acids Ind Eng Chem Res 2004 43 475 485 10.1021/ie0302239 Tong XD Xue B Sun Y A novel magnetic affinity support for protein adsorption and purification Biotechnol Prog 2001 17 134 139 11170491 10.1021/bp000134g Cocker TM Fee CJ Evans RA Preparation of magnetically susceptible polyacrylamide/magnetite beads for use in magnetically stabilized fluidized bed chromatography Biotechnol Bioeng 1997 53 79 87 10.1002/(SICI)1097-0290(19970105)53:1<79::AID-BIT11>3.3.CO;2-7 Chetty AS Gabis DH Burns MA Overcoming support limitations in magnetically stabilized fluidized bed separations Powder Technol 1991 64 165 174 10.1016/0032-5910(91)80017-D Arica Y Yavuz H Patir S Denizli A Immobilization of glucoamylase onto spacer-arm attached magnetic poly(methylmethacrylate) microspheres: characterization and application to a continuous flow reactor J Mol Catal B 2000 11 127 138 10.1016/S1381-1177(00)00223-X Akgöl S Denizli A Novel metal-chelate affinity sorbents for reversible use in catalase adsorption J Mol Catal B 2004 28 7 14 Odabasi M Denizli A Polyhydroxyethylmethacrylate based magnetic DNA-affinity beads for anti-DNA antibody removal from systemic lupus erythematosus patient plasma J Chromatogr B 2001 760 137 148 10.1016/S0378-4347(01)00264-X Denizli A Say R Piskin E Removal of aluminium by alizarin yellow attached magnetic poly(2-hydroxyethyl methacrylate) beads React Funct Polym 2003 55 99 107 10.1016/S1381-5148(02)00219-5 Tong XD Sun Y Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption Biotechnol Prog 2003 19 1721 1727 14656147 10.1021/bp030028p EEC-European Economic Community Resolution 80/177 Official J Eur Comm Directives CEE 80/777 1980 WHO, Directive de qualite pour l'eau de boisson Criteres d'hygiene et documentation a l'appui, Geneve 1986 2 254 Odabasi M Denizli A Cibacron Blue F3GA-attached magnetic poly(2-hydroxyethyl methacrylate) beads for human serum albumin adsorption Polym Int 2004 53 332 338 10.1002/pi.1305 Odabasi M Denizli A Cibacron Blue F3GA incorporated magnetic poly(2-hydroxyethyl methacrylate) beads for lysozyme adsorption J Appl Polym Sci 2004 93 719 725 10.1002/app.20485 Odabasi M Uzun L Denizli A Porous magnetic chelator support for albumin adsorption by immobilized metal affinity separation J Appl Polym Sci 2004 93 2501 2510 10.1002/app.20826 Akgöl S Türkmen D Denizli A Cu(II)-incorporated, histidine-containing, magnetic-metal-complexing beads as specific sorbents for the metal chelate affinity of albumin J Appl Polym Sci 2004 93 2669 2677 10.1002/app.20757 Özkara S Akgöl S Çanak Y Denizli A A novel magnetic adsorbent for immunoglobulin-G purification in magnetically stabilized fluidized bed Biotechnol Prog 2004

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==== Front Harm Reduct JHarm Reduction Journal1477-7517BioMed Central London 1477-7517-1-81533313010.1186/1477-7517-1-8ResearchHow do drug users define their progress in harm reduction programs? Qualitative research to develop user-generated outcomes Ruefli Terry [email protected] Susan J [email protected] New York Harm Reduction Educators, Inc (NYHRE), 903 Dawson St., Bronx, New York 104592 Academy for Educational Development (AED), 100 Fifth Ave., New York, New York 100112004 26 8 2004 1 8 8 8 2 2004 26 8 2004 Copyright © 2004 Ruefli and Rogers; licensee BioMed Central Ltd.2004Ruefli and Rogers; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Harm reduction is a relatively new and controversial model for treating drug users, with little formal research on its operation and effectiveness. In order to advance the study of harm reduction programs and our understanding of how drug users define their progress, qualitative research was conducted to develop outcomes of harm reduction programming that are culturally relevant, incremental, (i.e., capable of measuring change), and hierarchical (i.e., capable of showing how clients improve over time). Methods The study used nominal group technique (NGT) to develop the outcomes (phase 1) and focus group interviews to help validate the findings (phase 2). Study participants were recruited from a large harm-reduction program in New York City and involved approximately 120 clients in 10 groups in phase 1 and 120 clients in 10 focus groups in phase 2. Results Outcomes of 10 life areas important to drug users were developed that included between 10 to 15 incremental measures per outcome. The outcomes included ways of 1) making money; 2) getting something good to eat; 3) being housed/homeless; 4) relating to families; 5) getting needed programs/benefits/services; 6) handling health problems; 7) handling negative emotions; 8) handling legal problems; 9) improving oneself; and 10) handling drug-use problems. Findings also provided insights into drug users' lives and values, as well as a window into understanding how this population envisions a better quality of life. Results challenged traditional ways of measuring drug users based solely on quantity used and frequency of use. They suggest that more appropriate measures are based on the extent to which drug users organize their lives around drug use and how much drug use is integrated into their lives and negatively impacts other aspects of their lives. Conclusions Harm reduction and other programs serving active drug users and other marginalized people should not rely on institutionalized, provider-defined solutions to problems in living faced by their clients. ==== Body Background Harm reduction programs operate with the assumption that some people who engage in high-risk behaviors are unwilling or unable to abstain. Using a "low-threshold approach," they do not require that clients abstain from drug use in order to gain access to services, nor expect adherence to one service to be eligible for another. Rather than having abstinence goals set for them, clients in such programs take part in a goal-setting process, an approach that has been shown to correlate consistently with retention and success [1-3]. Providers help clients make connections among their complex attitudes, behaviors, and the change they are trying to pursue as a result of an interactive process – not a dogmatic format. Behavior change is regarded as incremental and based on the premise that people are more likely to initiate and maintain behavior changes if they have the power both to shape behavioral goals and enact them. Research on harm-reduction/syringe-exchange programs has been limited largely to demonstrating their success with reducing the transmission of HIV/AIDS among drug users [4-11]. While this is an important accomplishment, little is known about their impact in assisting drug users in making changes in life conditions, circumstances, and quality of life. This is partially because few efforts have been made to establish appropriate measures of client and program progress in these areas. The traditional field of drug treatment has generated many assessment tools including the Addiction Severity Index used extensively for treatment planning and outcome evaluation [12]. This tool, and others like it, such as the Chemical Dependency Assessment Profile (CDAP) [13] and the assessment forms created at the Institute of Behavioral Research at Texas Christian University (TCU/DATAR), generate severity ratings that are subjective ratings of the client's need for treatment derived by the clinician. The ASI interview asks questions related to domains or "problem areas" in substance abusing patients that have been determined by clinicians, not the patients themselves. Thus, despite the formally established validity and reliability of the tool, and others like it, the measures are developed from the perspective of the clinician and researcher and are designed to generate information that is consistent with their view of the world, not the world of drug users. Given the tenets of harm reduction in which drug users participate in their own goal setting, such tools lack cultural sensitivity and relevance. Denning's work on harm reduction psychotherapy (2000) is considerably more grounded in the life circumstances of drug users. Her Multidisciplinary Assessment Profile (MAP), a baseline assessment tool to use with chemical dependency clients, however, was not designed to generate information about what drug users consider to be realistic goals and progress towards these goals. If service providers are to guide an effective interactive process of goal setting, it is important that they understand the parameters of realistic, incremental behavior change from the perspective of the client. Since the development of the ASI, there has been growing movement to acknowledge the value of participatory research in which the "subjects" of research become directly involved with shaping the research agenda and designing data collection tools. Such an approach empowers the community participating in the research so that members are not objects acted upon but rather partners in an endeavor to improve their circumstances. This approach increases the cultural appropriateness of the way the research is conducted, the potential validity and reliability of the data that are generated, and the utility of the results. In order to advance the study of harm reduction programs and our understanding of how drug users define their own progress, we conducted participatory research to develop outcomes of harm reduction programming. The goal of the research was to involve program clients in a process that would generate valid measures that are 1) culturally relevant to the way they see the world and live their lives; 2) incremental – i.e., capable of measuring small changes, and 3) hierarchical – i.e., capable of showing how clients improve over time. This article summarizes information on the research methods used and the outcomes that were generated. Methods The research study was conducted in two phases. In the first phase, drug users participated in a group process using nominal group technique (NGT) [15] to develop the outcome measures. In the second phase, other drug users participated in focus group interviews to reflect on the measures developed and their validity for the drug-using population. Below, the sample and methods of the two phases are described in more detail. Sample Study participants were recruited from the New York Harm Reduction Educators, Inc. program, which has delivered comprehensive services to over 40,000 enrollees at six sites located in East Harlem and the Bronx, New York. To involve recipients in the study, clients were recruited by program staff and given a $10 incentive for participation. The study was advertised at the main program site, and a stratified convenience sample of approximately 120 clients was recruited for phase one of the study, and 120 for phase two, with some duplicate count of clients who participated in both phases of research. The sample was stratified by neighborhood, by time in the harm-reduction program, and by whether participants took part in the syringe exchange only or accessed a fuller range of services. The demographics of the sample closely represented the larger program and included 26 % African American, 50% Latino, and 24% white; 72% male and 28% female; 17% ≤ 29 years of age; 36% between 30–39, and 46% ≥ 40 years of age. Methodology In the first phase of the research, a facilitator independent of the program used NGT with 10 groups of approximately 12 individuals per group. This group process provides a structure for small group meetings so that client participation is maximized and judgments effectively pooled. The technique was especially helpful in establishing priorities in that it neutralized differences in status and verbal dominance among participants. Before the present study, NGT was used successfully with clients in the program to identify areas of life functioning that people like themselves (i.e., drug users) deemed to be the most important and meaningful in their lives. The top 10 "life" areas were money (income); housing; food (nutrition); family relations; self-improvement; connectedness to services/benefits/programs; dealing with negative feelings (mental health); health problems (physical health); and legal and drug use problems. To generate items in a scale for every outcome listed above, 12 people were recruited for a group that used several NGT process steps. First, the facilitator asked the group to contemplate a question related to the outcome of interest. For example, "What ways do people in your circumstance make money?" was asked to generate the outcome of source of income; "What ways or places do people in your circumstance get something good to eat?" was asked to generate the outcome of source of nutrition; and "What are the types of places that people in your circumstance live?" was asked to generate the outcome of housing. Next, the members of the group brainstormed their answer(s) to the question posed to them and the facilitator recorded these answers on a large chart. The facilitator continued to call upon the members until all ideas were recorded. Next, all group members received a packet of 10 index cards numbered 1 to 10. The facilitator engaged the group in ranking the ideas according to every individual's order of preference. This step started with the facilitator asking individuals to take out the index card marked #1. She then asked every group member to select from the list that idea that he/she considered the best (i.e., the best way to make money, the best way/place to get something good to eat, the best place to live, etc.) and write it down on card #1. She then asked everyone to take out card #10 and select the idea that he/she considered the worst (i.e., the worst way to make money, the worst way/place to get something good to eat, the worst place to live, etc.) and write it down on card #10. Finally, she led them through a similar ranking process with cards #2 – #5, used by the individuals to record their "next best" preferences and cards #6 – #9 to record their "next worst" preferences. This group work resulted in 10 scaled outcomes. In the second part of the research, 10 focus group interviews were conducted to allow more of the target population to reflect on the validity of the measures and further explore the meanings of the scaled items based on the lives of drug users. In most cases, a completely different group of drug users who had participated in the NGT process for a certain outcome were selected to participate in the focus group related to that same outcome. Data Analysis The analysis of the data from each NGT group for every outcome was done by first eliminating all ideas that received no votes. The remaining data were then analyzed by: 1) determining the total score for all remaining ideas (the individual score of each idea was based on the card number on which it was recorded – i.e., 1–10 – and the total was the sum of individual scores); 2) determining the mean score for every idea (dividing the total score for an idea by the total number of votes – i.e., cards received for the idea; 3) rank-ordering the ideas by mean score; and 4) grouping the 40 or more individual ideas with similar mean scores by larger concepts so that every outcome had about 12 hierarchical, scaled items from best to worst, with mean scores from low to high. Results The results of the preliminary research are displayed in Table 1 (see Additional file: 1) showing every individual outcome and the hierarchical scale of items that measure incremental change from better to worse. "Better" items in every outcome, near the top of the scale, were those items that had received a mean score of 5 or better while "worse" items, near the bottom of the scale, were those items that received a mean score of 6 or higher. A summary of results for every outcome is presented below. Money (income) The outcome 'money (income)' includes a hierarchical scale of 11 items measuring better to worse 'ways of making money'. According to the clients, better ways of making money were from entitlements; a legitimate job; from family (i.e., through marriage, inheritance); or borrowing from others. Worse ways of making money were from hustling (i.e., conning or informing to police); stealing (i.e.,"boosting" – stealing); drug trade (e.g., selling, holding, transporting); panhandling; more serious criminal activity (i.e., credit card fraud, robbery, hitman); sex work; and selling blood or body parts. When other program clients reviewed this outcome scale, they felt it reflected their lives overall. One exception was that many felt that "job" (employment, peddling, odd jobs, volunteer) should be at the top of the scale rather than "entitlements." Some saw entitlements as "an easy way to make money – "it's a way of survival," – while having a job offered more independence and feelings of self-esteem. Other changes suggested by individuals were that "stealing," "drug trade" and "hustling, police informant" should be further down on the scale while "panhandling" should be further up. Strong negative feelings were expressed about being a police informant;" "Where I come from, when out on the street..., you don't inform the police! Police will lie to you and use you, then throw you back to the dogs and you're dead." Focus group participants spoke about what money meant to them and what life was like when one made money via drug trade, sex work or selling blood or body parts. Like most people, they felt having money offered a sense of freedom and independence. In addition, many felt that having it contributed to self-esteem and allowed you to help others you cared for: "It lets you help a family member who needs help"; "It helps support my spouse... and give some money to my son when I can." Regarding money making from drug trade, study participants felt, overall, that it was unstable and risky. It's like addiction. It's an adrenaline pump ... that keeps you in it for so long. You do it to keep your habit going... If you sell what you use, it's not good. You wind up doing all the drugs, then you have to run for your life from the dealer. It's an unstable life...and the consequences are great if you get caught. You go to jail, get clean, come back out, and start all over again. It's a never-ending cycle.... Eventually your luck runs out and you either go to jail or get killed. Sex work was also described in negative terms: That's the last thing you want to have to do, man or woman. Selling your body is the worst thing you could do for yourself. You can get HIV, hepatitis C, or even get killed. You don't know who this person is that you are engaging in this sex at with. He could be a serial killer. Selling blood and body parts were described as both limited and, again, risky: Now you are put on a computer and once they have a record of your blood test, you can't do it because they can screen you from sight to sight... If you sell a body part, they might not wait for you to die to get it, they might hunt you down and kill you. Housing The outcome 'housing' is made up of a hierarchical scale of 11 items measuring the better to worse places to live. Based on client input, better places to live were a house or apartment that you rented or owned; a friend's home; a drug program; a family member's home; housing provided through a social program; institutionalized housing, such as a shelter or hospital; and homelessness that was considered "least severe (e.g., sleeping on the subways or at a bus station). Clients considered worse places to live to be jail, homelessness considered to represent an intermediate level of severity (sleeping in cars, bathrooms, hallways, abandoned buildings) or the most severe level (in tunnels, caves, sewers, parks, on a roof). When other program clients reviewed this outcome scale they agreed, overall, that it reflected the reality of their lives. A few suggested minor changes in the scale could be made such as moving "jail" as a housing option to the very end of the scale because, as one said, "I don't want my freedom taken away – it's degrading and lowers your self-esteem being subject to a strip search at any time." A few others suggested putting "apt/room that you rent or own" before "friend's home." Most felt that it was better to have your own place than to live with family or friends because "it makes you feel independent, feel human – it's an accomplishment." They also offered some insight on what it was like being a drug user and living with a family member vs. a friend. A family member would let you get away with more than a friend would. With a friend, you would have to be on time, with all of your part of the rent money, food money, and clean up after yourself. With family you might dib and dab a little with the rent money or get out of doing some things around the house. However, when you're on drugs, going to your family is not good because they can give you the boot. Other "windows" of insight into how drug users deal with housing came in participants' discussion of being in a drug program, institutionalized housing (shelters, hotels, hospitals, SROs), and living on the street. Participants thought that drug programs offered an important option for housing but also saw them as a last resort: They give you structure and are stable – you can get food, clothes and confidence. Last resort when you have no place to stay and have no money... It's the place to go if you want to change your life around. Some program clients saw institutionalized housing as a crutch, while others discussed the advantages and disadvantages of various types of institutionalized housing: I became "shelterized" after being in a shelter for several years. I got locked into a routine where you don't want to take care of yourself because the basics are provided for you. Life was sweet, too sweet – I had no responsibility. Some hotels, depending on where they are located, have a high level of theft with no security. Hospitals will take you in depending on the weather and your state of mind. The homeless quarters and the psyc ward are often connected. Sometimes you have to fake it, like you need psychiatric care, if you want to get off the street for a while. If you act like you are going to harm yourself, they give you a bed fast. SROs are like apartments but they have rules... Some don't allow company but others do. You have to sign visitors in and out at the desk. Tier 1 and 2 housing is for families. But it's only temporary – 30 to 60 days. Then you have to pack up everything and move somewhere else. Section 8 housing has a lot of limitations. You have to be a family that is homeless living in a shelter, a victim of domestic violence, or in the witness-protection program. You can't have any felonies on your record. Some of section 8 is only available if you are HIV positive. Generally, clients felt that living on the street was a last resort but an option that could work: If you bum everything out by not following rules, stealing, looking for fights or taking drugs, then the street will become your home... But you can make the street work for you if you know how to survive. I did. You pick your area and take a claim for it. I had a half car that was my roof... I even evicted some people from my neighborhood because they didn't act right. We had our own rules. Food (nutrition) The outcome 'food (nutrition)' is made up a hierarchical scale of 11 items measuring the better to worse 'ways/places to get something good to eat'. According to the clients, better ways/places to get something good to eat were to cook food yourself; from friends or family; from a supermarket; from place that gave out free food (from soup kitchen, shelter, pantry, social gathering); buying food (with food stamps or from money earned); or from a restaurant. Clients considered worse ways/places of getting food were from begging or stealing; from institutions (hospital, jail), from trying to provide for yourself (hunting, fishing); and, lastly, from the garbage. After other program clients reviewed the developed scale, they generally agreed that it reflected their lives. The exceptions were that some felt that "buying food" should be placed higher in the scale, preferably at the top. The rationale was that before you could cook food yourself, you needed to buy it. In addition, others felt that "food from facilities" and "providing your own food" should be placed before stealing food in that stealing food involved risk and possible repercussions. Study participants spoke about what they considered "something good to eat." They most often mentioned that food needed to be tasteful, although not necessarily nutritious. The feelings that they associated with getting something good to eat were "feeling good," "wanting to act civil," and "wanting to treat people better." Feelings associated with not getting something good to eat were "feeling cheated" and "developing an instant attitude." They also discussed why "cooking food yourself" was higher on the scale than "going to a restaurant" or having others prepare the food for you. The importance of self-sufficiency emerged when participants spoke about the value of "cooking it the way you want it" and the feeling of comfort that came from "doing it yourself" and being able to "be at home with my girl and be able to afford a full-course meal." Participants had much to say about the topic of "free food" and the better/worse places of getting. Although "the price was right," and they were all aware of "street sheets" listing several places to go for free food, they also spoke about traveling long distances, waiting on long lines, walking up several flights of stairs, and having to have a referral and register with a program to get food. The factors that affected their decision about where to get food were the attitudes of the staff, the quality of food offered (i.e., brand names were preferred over generic, U.S.D.A. – grade foods), and whether the program also offered other needed services (e.g., some pantries offered services like showers and laundry facilities). Some participants contended that some program staff in pantries "pick through the groceries and bag up the best stuff for themselves and friends." Other revealing insights that drug users had about food were that they did not feel it was ever necessary to steal or beg for food: "There are plenty of places to get food. Anyone you see stealing food or begging is doing it for a profit, to be able to purchase something else." A number of participants referred to "dumpster divers" (people who eat street or building garbage) as people who were mentally ill and took great risk of eating contaminated food. Most felt more comfortable eating leftover, pre-wrapped food from fast-food restaurants than resorting to street dumpsters. Family relations The outcome family relations includes a hierarchical scale of 15 items measuring the better to worse 'types of family relations'. At the top of the scale clients considered better ways of relating to family to include loving your family; taking part in special family gatherings; having positive communication (open, honest, tolerant); interacting directly with members (playing games, picnics, talking about family history); arguing; showing support and respect, spending high quality time; and engaging in passive contact (movies, TV, reading the Bible). Clients felt the worse ways of relating to family were showing a lack of respect between members (being stigmatized/disrespected for who you are, talking about drugs around children); members' having negative attitudes toward one another (envy, judgment, alienation); conflicting lifestyles; engaging in abusive relations (incest, sexual abuse, violence); having difficulties with financial support; abandoning a family member; or being deceitful (stealing, gossiping, lying). Upon reflecting on the scaled outcome later, other program clients generally agreed that the scale reflected their lives, with a few exceptions. Several felt that "arguing," in the middle of the scale, should be placed further down on the scale as they saw it as a way of relating that often leads to abuse. In addition, there was some disagreement on the order of the items considered the worse types of family relations. Some clients felt that "abusive relations" should be listed last. These individuals spoke painfully about how abusive relations in their childhood had damaged them throughout life, and others spoke about how exposure of the abuse within their family had created lasting division between members. Client input on the items "love," "respect," and "negative attitudes" illuminates the meaning of these terms in the lives of drug users. "Love" was seen as a building block and foundation for family relations. It was equated with respect among family members, with one client asserting that "Love for my family may mean not spending time with them so that I do not expose them to my drug use." Clients felt that indicators of family respect were listening, letting people have their say, giving people the benefit of the doubt, and living life your way without interfering with others. Clients reflected on the negative attitudes they had experienced around family members. Along with outwardly judgmental remarks, clients also experienced a great deal of nonverbal behavior that they interpreted as negative attitudes. Examples included when they walked into the room where family members were conversing, and people suddenly stopped speaking or hid their purses. Overall, clients felt that a family member's drug use should not necessarily engender negative attitudes among other family members and that their families needed to learn more about understanding the harm reduction approach. Connection to services, programs and benefits The outcome 'connection to services, programs and benefits' consists of a hierarchical scale of 12 items measuring the better to worse 'types of services/ programs/benefits available to drug users'. Clients felt better types of services to connect with were those related to housing; HIV/AIDS assistance; mental health; drug treatment; entitlements (i.e., public assistance, SSI, and social services); and harm reduction (outreach, needle exchange, condoms). They considered the less preferred (i.e., worse) available services to be those in mainstream institutions (churches, library, legal services); "getting-connected" services (escort services, resource directories); support services (12 step, women's groups); family-prevention services (parenting skills, domestic violence services); stress reduction (acupuncture, field trips); and work (WEP) programs. Later, when other clients reviewed the developed outcome, most felt that it overall reflected their lives. The one change that a sizable number of clients called for was to put harm reduction services farther up on the scale. The value they placed on this type of service was shown in a number of comments: Harm reduction has taught us a lot about taking care of yourself physically, mentally and emotionally. If you are using drugs, it teaches you how to use drugs safely and in a safe environment. If you want to stop using, there are places to go to get the help you need. If you are out on the street hustling, selling your body it teaches you about using protection. Harm reduction is very important because it taught me a lot about how to take care of myself, manage my drug use, use my needles properly, and reduce my stress. In addition, certain individuals, based on their circumstances, made other suggested changes. One client who disclosed himself as HIV sero-positive said that "AIDS-related services" was the best service on the list for him. Another client remarked that "drug treatment" would need to be listed before housing since you are required to be drug-free to get housing. Still another felt that all the listed services were important "because they can assist me in preparing for my future." The clients discussed why "support services" (12-step programs, advocacy groups) were fairly far down on the scale. Overall, they felt this was because participation in these programs was dependent on giving up drugs, which some people are not ready to do. They also felt some people do not agree with the philosophy of the programs nor are they ready to be in a group environment. Clients also spoke at length about why the "WEP (Work Related) program" was listed last on the scale. Although they thought it might benefit people who have no skills, they felt, overall, that the program was degrading. For those who may have skills, it's kind of degrading in a way because you are working for a check that you are receiving from public assistance. For a single person you might get $68.00 every 2 weeks and you are doing the same kind of work that the people earning above minimum wage are getting. You can be working in the Parks department, cleaning people's toilets or picking up paper in the street for the sanitation dept. Some of it can be real degrading and discouraging. Self-improvement The outcome 'self-improvement' consists of a hierarchical scale of 12 items measuring the better to worse 'ways of improving yourself'. Study participants felt that better ways of improving yourself were having a better relationship with yourself (self-love, respect) and with others; getting and staying clean from drugs; being spiritual; taking part in self-help groups (12-step programs, support groups); working or developing work skills; and engaging in stress-reduction activities. They considered less preferred or "worse" ways of improving yourself to be helping others; taking care of yourself (i.e., going to dentist, taking medications, dieting, going to gym); being more responsible (i.e., living on a budget, accomplishing goals); behaving yourself (stop lying, stay out of trouble); and having a hobby (i.e., art work, boating, fishing, hunting). After a different group of program clients had reviewed this outcome, most felt that it adequately reflected their lives. A few individuals suggested that "caring for self" and "being more responsible" (i.e., items #9 and #10) should be listed further up on the scale. In addition, one individual felt that "becoming more spiritual" should be first on the scale, "because if you have God in your life, everything else will fall into place." Program clients were asked about the meaning of "self-improvement," "self-respect," "relating to others" and other items as they appeared on the scale. Concerning self-improvement, clients often thought of the topic as one that involved personal goal attainment. Setting goals that are positive and reaching them. Then setting another and reaching it, one step at a time.... Setting up a network that will help me to build a foundation of positive aspects in my life that I can follow. The clients described self-respect as requiring self-esteem, as being linked with showing respect for others and with how you physically appear to others, and as dependent on managing your drug use. If you have self-esteem and care for yourself..., respect will come. By you respecting yourself and wanting to be treated a certain way, you know you have to respect others to get it back in return. If I looked better, I would feel better about myself. A lot of time when people are drugging, they get caught up in a lot of things and before they know it, they have done some things that have cost them their self-respect, so getting it back is important to be able to get on with your life. In clients' discussion of the meaning of "relating better to others," several indicators emerged such as honest communication; holding an intelligent conversation about yourself; being comfortable relating your feeling to others; and listening. Their thoughts on "getting/staying clean" (item #3) demonstrated the challenges they face and the degree to which their lives must change to stay clean. It was a hard process for me because I would always fool myself that it wasn't the right time.... You can't do it for someone else, it has to be for you. It took me becoming homeless to decide that I had to make some changes in my life. Now that I have a new apartment, I want to keep it. My budget won't allow me to get high and keep my rent paid. Once I got out (of jail), my body was clean but my mind was still dirty. Mentally I still wanted to do drugs... I had to leave people, places and things alone because I feel powerless over the influence of others. Being around positive people and getting the support of groups helped me stay clean. Clients also provided rich detail on what they meant by "behaving myself" (item #11), including this response: It's the whole package. Your attitude, the way you talk, the language you use. When you start to change your life for the better, everything changes. You don't use a lot of 4-letter words. You want to socialize with different people in a different atmosphere. Not getting high where you work at; being more responsible. Finally, the clients were asked why "working/developing work skills" (item #6) was as far down on the scale as it was. Most acknowledged that this was a goal that many drug users are not yet ready to achieve, given their difficulty functioning in an environment that they are not used to. If you are coming into work and you are in this other world where you are not sick (to others), but you are not well either, it is hard to function. You have to have a functional mind that is able to concentrate on work...and for a lot of people, they are not there yet. Alternatively, their discussion suggested that volunteering was a better way to approach the world of work: "I started volunteering here at NYHRE, and I intend to go to computer school so I can get a better job." Mental health The outcome 'mental health' consists of a hierarchical scale of 13 items measuring the better to worse 'ways of handling negative feelings'. Study participants felt that better ways of handling negative feelings were getting informal support; (from friends, support groups), spiritual help (going to church, praying); or professional help (from a doctor, counselor); working; engaging in diversions (interacting with children; going to ball game or the beach, singing), or in stress reduction (smoking, massage, sex) and physical activities (exercise, cooking, sports). They considered worse ways of dealing with negative feelings to be engaging in violence against self (suicide, bulimia, anorexia); outward violence (hurting others, breaking things); bringing negative feelings into social relations (into marriage, when visiting someone in jail); withdrawing ("isolating"); and engaging in illicit activity (working the streets, using drugs, gambling). When another group of program clients reviewed the scaled outcome, they saw the relevance of all the items and agreed on the general order of the items in the scale. A few clients suggested some minor adjustments in the scale, however. For example, a few felt that "professional help" (item #3) should be considered the best way of handling negative feelings, rather than "get support" (from friends, support groups). For the most part, however, the majority of the clients agreed that getting support from friends and support groups was more functional for people in their circumstance than going to a professional because of issues of availability. As one person put it, "The drug man never sleeps" and people involved in this culture need easy access to those who can help them with their negative feelings. When you are out in the street drinking and drugging, there is something going on at every corner 24 hours of the day. Support from friends and groups are available to you on those off hours when "professional help" is not. This was also the rationale for few clients as to why "spiritual help" should be placed before professional help – you can pray at any time. Other individual clients felt that "social relationships" should be further up on the scale because peers and loved ones were often the most understanding. You need communication with someone that understands you and is willing to put up with your shortcomings. Problems do arise if one gets high and the other does not, but you can usually work this out. Things get tough sometimes but she helps to keep the balance in the relationship. My spouse helped me with my addiction. In regard to how drug users resorted to abuse in deal with negative feelings, the clients often referred to circumstances involving drugs. When verbal abuse did not work, they often resorted to physical abuse. A spouse will be abused when you want your drugs and you don't have the money. You know that she has the money but she won't give it to you. When selling drugs and someone comes to you with short money, even if it is only $1, he might get his butt whipped. Dealing with health problems The outcome 'dealing with health problems' consists of a hierarchical scale of 12 items measuring the better to worse 'ways of handling health problems'. The clients in the study considered better ways of handling health problems to be using home remedies (external and internal cleansing, praying); stress reduction; drug treatment/therapy; "clean living" (i.e., reduced drug use, taking meds, stopping smoking); seeing the doctor; and getting health screenings. They felt less preferred (i.e., worse) ways of dealing with health problems were maintaining a good diet, getting health education information, exercising, using alternative therapies (i.e., fasting, herbs, psychic readings, witch doctors), exhibiting negative emotions (depression, denial, suicide, anger); and using illicit drugs. After another group of program clients reviewed this outcome scale, they generally felt that it reflected their lives, with a few exceptions. Several felt that "see a doctor," "educate yourself" and "alternative therapies" should be higher on the scale. Most clients felt that "home remedies" should stay at the top of the scale because "they work the best." When they spoke about their experiences with doctors, it often was not positive. The waiting is horrible. As an inpatient, you could die before you see a doctor. Once you are identified as an addict, whether on methadone or still using drugs, you're discriminated against. Sometimes I am too leery to go to see a doctor. I may wait for someone else to go to the doctor first and then get their opinion. When asked about the health problems they encountered, client usually mentioned serious conditions (cancer, STDs, HIV, pneumonia), indicating that ailments were not a health program unless they had become serious. Regarding drug treatment, clients saw it as a positive way to deal with health problems, with certain parameters. "Drug treatment is not going to help you if you are not ready to stop using... It won't help you unless you have a follow-up plan like a support network at a church, family or groups, and being around positive people." Clients also discussed how other items on the scale were related to their drug use. Several felt that "using illegal drugs" should be at the very bottom of the scale, but opinions about this varied based on level of drug use. Clients knew that drugs could eventually bring about bad health but were often so out of touch with their feelings while doing drugs that they thought they were healthy: When I was on a constant run (doing drugs), I didn't get sick. Thought I had a wonder drug. Didn't feel anything; drugs preserved me. I didn't get headaches, toothaches or colds. If I was sick, the drugs controlled my inner body, I couldn't feel a thing. They felt that the item on the scale "educate yourself about health" was especially important for drug users who are often controlled by their substance: No one used to take vitamins because your drug controlled your mind. You couldn't eat properly because you had to get high first. Education about my health has helped me make some changes. Before I didn't go to a doctor. Now I make an effort to go on a regular basis. Dealing with drug use problems The outcome 'dealing with drug-use problems' consists of a hierarchical scale of 17 items measuring the better to worse 'ways of handling drug-use problems'. Study participants felt that better ways to handle drug-use problems were to admit the problem (and make amends with family); engage in religious activity (go to church, pray); get social support (from support groups, asking for help, making new positive friends); go into drug treatment; quit using drugs; get professional help (therapy, education about drugs, medications); stay distracted (keep busy, play with kids); and avoid the drug culture (avoid places that trigger drug use, drug paraphernalia). Clients considered the less helpful (i.e., worse) ways of handling drug-use problems were to follow a treatment plan (go to the hospital, take and not sell medications), get family support or spiritual guidance (from 12-step programs, minister); be in jail; be honest with yourself (reflect on past behaviors and pain associated with use); be deceitful (lie, manipulate others); engage in illegal activity (i.e., deal drugs, steal, prostitution); "isolate"; and continue to binge. After reviewing the developed outcome, another group of program clients generally agreed that the scale reflected their lives on the better to worse ways of handling drug-use problems. Several people commented that the items that were near the bottom of list, or the worse ways of handling drug use, were not ways of handling the problem but were, in fact, the kinds of things that went on when your drug use was out of control. They described in graphic terms what this looked like: To be focused every minute of every day on just getting the next bag of dope. My life is non-functional... I am a zombie. Wake up in the morning, get dressed and head straight for the corner to hustle up enough money to get that bag of dope. Binging is like being on a mission. You go all the way out until everything is gone.... It can be one hour, a day or longer. It is when you have used all your resources and there is no more to be had. There is no one left for you to use or manipulate. They also talked about why it was important to handle problems with drug use. One person admitted, "Your drug use is like a marriage, something you live with for life," and several clients talked about what their life looked like when they were able to handle their drug-use problems. I need to have something with structure in my life to keep going... so you can function better... go forward... handle you apartment, raise your kids, keep yourself clean... stay out of jail and live a longer life. My everyday life is my life now. The clients made several other insightful remarks about various items in this outcome. They commented on how "praying" helped them to function: "Praying helps me get things straight in my head." "It makes me strong and gives me more confidence,"; "Praying makes me more humble"; and "When I pray I feel more positive in my thinking." Regarding the item "follow a treatment plan," some people felt it was farther down on the scale because of the coercive aspect they associated with it: "Sometimes following a treatment plan is what you have to do because you have to see your parole officer every week, so you are forced to do it." In describing their experience in jail, many felt it did not help with problems with their drug use because it is very easy to get drugs in jail. The clients did feel, however, that it was something to be avoided at all costs: What you experience in jail makes you never want to go through that again. It takes your freedom away. It changed me. Now I don't even steal a Hershey bar. Dealing with legal problems The outcome 'dealing with legal problems' consists of a hierarchical scale of 11 items measuring the better to worse 'ways of handling legal problems'. The participants in the study considered better ways to handle legal problems were to pay, go see, and speak with a legal professional; address the problem yourself (go to law library, represent yourself, write to the judge); speak to a non-legal person (employer, counselor, parole officer, case manager); respect the law (by serving time, making court appearances); and learn from mistakes. The clients in the study considered worse ways of dealing with legal problems to be disrespecting the law (breaking the law, not respecting authority); facing the consequences of one's actions (serve time in prison or drug program, give up parental rights); avoiding legal responsibility (run from parole, leave the state, not show up in court, jump bail, ignore bench warrants); and relying on support from friends. When a different group of program clients reviewed the outcome they agreed overall with the order of the items in the scale. They offered rich detail on specific items on the scale and insight into how drug users experience legal problems. Drug users confront a wide variety of legal problems, including being arrested for various drug-related charges; police harassment related to petty crimes like loitering or suspicion of a crime; legal problems related to one's children and the Bureau of Child Welfare; and taking part in a hearing to qualify for SSI. Clients talked about their experience with legal professionals and items near the top of the scale. Many agreed that it was best when you could pay for an attorney, or, as one client put it, "Money talks and bullshit walks." However, they also realized that the steps in dealing with a professional first involved seeing and talking to one to find out the fee for services. Clients had varied experiences with professional attorneys, with several agreeing that legal aid lawyers were most helpful. I prefer legal aid lawyers because they work from the heart and not by what you put in their pockets. In housing I had a legal aid lawyer who helped me in a very positive way. A private, paid lawyer to help me keep my kids did not do what he was supposed to do. It was an SSI case and I had to pay and I got very little help or feedback from the lawyer at all. As with other outcomes that have been reviewed, the participants in the study spoke favorably about trying to solve the problem themselves (item #4 on the scale). It is good to do everything you can to help yourself first before you pay a legal professional or seek out their help. You might be homeless, out there on the streets...and ready to come in and get your life together.... You need to investigate how to clean up any legal problems that may be lingering. Sometimes friends may have gone through a similar experience and can tell you some of the steps they took to avoid jail or paying fines. Clients talked about how they "learned from legal mistakes" (item #7) and what "disrespect the law" (item #8) meant to them. Learning from legal mistakes often involved experiencing the consequences when the police caught up with you: I use to smoke my pipe out on the street and didn't care about the cops or anybody...When I saw the cops, I would run and hide and thought I got away from them. But when I came out of hiding, they were waiting for me and I got arrested. Clients associated several different acts with "disrespecting the law" and often spoke of "testing" the authorities by jumping the subway turnstile, going to the bathroom in the street, jay-walking, and cursing out the cops. Another interesting insight into the lives of drug users was how the clients felt that the law did not understand their ability to manage their drug use and lead a responsible life. Overall, they knew that when their drug use was out of control, they realized their respect for the law was "the farthest thing from your mind." One woman described how during a chaotic period she became suicidal and her children were taken away by the authorities. However, after she had effectively managed her drug use for some time and had sought out legal representation, even then she had not been allowed access to her children for the past six years. Discussion The methodology used in the study contributed to the field of harm reduction and how to work more effectively with drug users. Meaningful outcomes for active drug users cannot be accurately measured in "either/or" terms (i.e., drug use vs. abstinence) or reflect a yardstick of achievement that is not culturally based in the lives of program clients. In that harm reduction programs work with drug users "where they are" and strive for incremental change that can be realistically accomplished, the results of the study represent the field's first attempt to establish relevant, culturally sensitive outcomes for measuring client and program success. Rather than using measures/standards developed by researchers in a different cultural world, the generated outcomes seek to more closely represent the lives of the population we are trying to understand and serve. The ethics and benefits of this participatory research/evaluation approach have been acknowledged by many [16-19]. This said, it should be noted that that the major limitation of the research was that it was done with a representative sample of only one harm reduction program in an urban area. And, although the sample included a variety of program clients whose drug use was characterized on a continuum from stable to chaotic, the external validity of the measures may be questionable for use with harm reduction programs having a different client population. The methodology is one that can be used by other harm reduction programs to help them identify ranked goals in each of the life areas for their particular population. In addition to its methodological contribution, the study provided some important insights into drug users' lives and values, and an increased understanding of how this population envisions a better quality of life. Based on a number of items in the scaled outcomes, results showed that drug users often cited traditional measures in defining their life progress. Examples of this include having a legitimate job as a positive measure of source of income; being homeless and sleeping on the street as a measure of an undesirable housing situation; having an open and honest relationship with one's family as a measure of positive family relations; avoiding legal responsibility (i.e., jumping bail, running from parole) as a measure of an ineffective way of dealing with legal problems; and getting/staying clean of drugs as a positive measure of how to deal with drug use. It is generally felt that drug users' low ratings of certain activities as solutions to their problems (i.e., crime, prostitution) does not reflect a belief that these activities are inherently bad or immoral. Rather, these ratings reflect drug users' pragmatic nature and recognition that these activities can be impractical and dangerous. Other findings from the qualitative research were somewhat counterintuitive to those outside the drug-use culture and reflect the realities of poverty, racism, social isolation, past trauma, and discrimination faced by individuals in this stigmatized population. For example, the study sample considered the very best way of making money was through entitlements (i.e., welfare, disability). This finding reflects the fact that since drug users struggle for day-to-day survival and often have a criminal record, many do not have the confidence, skills or opportunities to make a legitimate living in the dominant culture. It also reflects the reality that getting needed resources via entitlements is relatively easy and dependable for drug users while getting pay from a part-time job can be problematic when an employer in the drug trade can disappear on pay day. The stark realities of the drug user world were apparent in the fact that sex work and selling blood or body parts were ways that some individuals survived. Findings related to the housing and nutrition outcomes also provided a window into the lives of many drug users. For example, being in jail was considered a more preferred housing arrangement than many forms of being homeless; eating out of the garbage – "dumpster diving" – was considered the worst way to try to get something good to eat. Interestingly, when it came to family relations, violence among family members was considered less problematic than being deceitful (stealing, gossip, lies) and may partially reflect the culture's shared value of openly expressing feelings. In regard to the outcome of connectedness to services and programs, it was not that surprising that the sample valued those services that addressed their day-to-day survival needs (entitlement benefits and programs related to housing, AIDS, and drug treatment), more than those that addressed less immediate needs (prevention, stress reduction, nutrition, and employment services). Certain consistencies across the outcomes and items in the scales shed light on other values of drug users and the barriers they face. Overall, the sample indicated that the most preferred way of living was one in which they could try to work things out for themselves and remain independent of the dominant culture. This was seen in such examples as "having your own place to live"; "cooking food yourself as a way of getting good food"; "developing a better relationship with self as a way of improving yourself"; "praying and getting support from friends in order to deal with negative feelings"; "relying on home remedies to address health problems"; "admitting you have a problem and making amends as a way of handling problems with drug use"; and "going to a law library and doing research as a way of dealing with legal problems." These findings are not surprising, considering the fact that drug users often experience stigma and discrimination when they try to rely on the traditional service delivery system and, as a result, remain isolated and marginalized. It is also a finding that challenges the dominant view of drug users as lazy, dependent, and not wanting to change. Implications for the field This preliminary research needs to be expanded in order to develop more valid and reliable outcome measures for the field of harm reduction. Other harm reduction programs are encouraged to conduct similar research with drug users in their locales and share their results. In addition, to validate that the outcomes are not simply research artifacts but reflective of the target population, field research can be conducted. For example, "hanging out" with the homeless and watching them as they spend the day looking for the best place to be homeless would help validate the outcome on housing. With measures that are science-based and evaluation results that can demonstrate the effectiveness of this approach to working with drug users, harm reduction programs will stand a better chance of receiving funding from potential donors. In addition to their use, these measures for program outcome evaluation can also benefit the clinical component of programs. Case managers could collaboratively use the methodology in sessions with individual clients to develop relevant and realistic treatment plans. Future plans for behavior change developed in one-on-one sessions and using user-generated measures of progress have more potential for achievement than plans that do not consider how clients define progress. Conclusion Clearly drug users have a set of workable solutions for meeting their own survival needs. Results of the present study show that they can often view institutions in the existing culture as irrelevant when addressing day-to-day living problems. The scales and rankings of many of the socially approved ways of solving life's problems show that just as there is an underground economy, there is a whole underground subculture where those marginalized from the mainstream have developed a culture with its own set of relevant structures, informal relationships, and home-grown recipes for addressing life's challenges. These study findings convey an understanding of drug users as people who are interested in positive change in all areas of their lives, and that by empowering them in a process to identify their own goals, they may be more motivated and engaged in the program. The findings also provide some initial empirical evidence that challenges traditional ways of measuring drug use based solely on quantity and frequency. Results suggest that more appropriate measures may be the extent to which the drug users organize their lives around drug use, the extent to which drug use is integrated into their lives, and the extent to which drug use negatively impacts other aspects of their lives. They also suggest that harm reduction and other programs serving active drug users and other marginalized people should not rely on institutionalized, provider-defined solutions to the problems in living faced by their clients. Rather, drug users should be assisted with problem solving by being encouraged to consult their own set of culturally shared solutions. Competing interests None declared. Authors' Contributions T.R. conceived the study, designed the research, and provided input on the manuscript. S.R. coordinated the study, provided oversight on data collection, analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript. Supplementary Material Additional file 1 Outcomes of Harm Reduction Programming to Measure Incremental Change from Better to Worse. This is a tabular form of all categories of behavior discussed in the paper. Click here for file Acknowledgements The authors wish to acknowledge several individuals who contributed to the research. First, we thank the clients of the NYHRE harm reduction program who took part in the research. Their patience and commitment to the group process was commendable and their honesty and directness appreciated. Thanks also go to two people at NYHRE who logistically set up of the research: Martha Hornsby, former Deputy Director and Eddie Rivera, Project Coordinator. We also appreciate the work of AED staff and consultants: Kathryne Leak and Sarah Anderson who conducted the groups with clients, Stacy Silverstein who assisted with the development of focus group protocol, Amy Richie who analyzed the data, Noemi Corujo who formatted the manuscript and Elayne Archer who edited it. Finally, we would like to thank Edith Springer and Ernie Drucker, PhD for their encouragement and input on this research. ==== Refs Ojehagen A Berglund M Changes in drinking goals in a two-year outpatient alcoholic treatment program Addictive Behaviors 1989 14 1 9 2718819 10.1016/0306-4603(89)90011-7 Sanchez-Craig M Lei H Disadvantages of imposing the goal of abstinence on problem drinkers: An empirical study British Journal of Addiction 1986 81 505 512 3463350 Sobell MB Sobell L Bogardis J Leo GI Skinner W Problem drinkers' perception of whether treatment goals should be self-selected or therapist-selected Behavior Therapy 1992 23 43 52 Kaplan R Heimer R HIV prevalence among intravenous drug users: model-based estimates from new Haven's legal needle exchange Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 1992 5 163 169 Lurie P Reingodl AL Bowser R Chen D Foley J Guydish J The public health impact of needle exchange programs in the United States and abroad San Francisco CA: University of California, Berkeley, School of Public Health, the Institute for Health Studies 1993 Heimer R Kaplan EH O'Keefe R Khoshnood K Altice F Three years of needle exchange in New Haven: what have we learned? AIDS and Public Policy Journal 1994 9 59 73 Watters J Estilo MJ Clark GL Lorvick J Syringe and needle exchange as HIV/AIDS prevention for injection drug users JAMA 1994 271 115 120 8264065 10.1001/jama.271.2.115 Hagan H Des Jarlais DC Friedman SR Purchase D Alter MJ Reduced risk of hepatitis B and hepatitis C among injecting drug users participating in the Tacoma syringe exchange program American Journal of Public Health 1995 85 1531 1537 7485666 Des Jarlais DC Marmon M Paone D Titas S Shi Q Perlis R Jose B Friedman SR HIV incidence among injecting drug users in New York City syringe-exchange programmes Lancet 1996 348 987 891 8855855 10.1016/S0140-6736(96)02536-6 Vlahov D Junge B The role of needle exchange programs in HIV prevention Public Health Rep 1998 113 75 80 9722812 Vlahov D Junge B Brookmeyer R Cohn S Riley E Armenian H Beilenson P Reductions in high risk drug use behaviors among participants in the Baltimore needle exchange program Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 1997 16 400 406 9420320 McLellan AT Luborsky L O'Brien CP Woody GE An improved diagnostic instrument for substance abuse patients: The Addiction Severity Index J Nerv Ment Dis 1980 168 26 33 7351540 Harrell TH Honaker LM Davis E Cognitive and behavior dimensions of dysfunction in alcohol and polydrug abusers Journal of Substance Abuse 1991 3 415 426 1821295 Denning P Practicing harm reduction psychotherapy: an alternative approach to addictions 2000 New York: Guilford Press Delbecq AL Van de Van DH Gustafson DH Group techniques for program planning: A guide to nominal group and Delphi processes 1976 Glenview, Ill: Scott, Foresman Fetterman DM Kaftarian SJ Wandersman A Eds Empowerment evaluation 1996 Thousand Oaks CA: Sage Publications Selener D Participatory action research and social change Ithaca NY: Cornell Participatory Action Research Network, Cornell University 1997 Hopson RK Ed How and why language matters in evaluation New Directions for Evaluation 2000 86 Ryan KE Destefano L Evaluation as a democratic process: Promoting inclusion, dialogue and deliberation New Directions for Evaluation 2000 86

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-431529402010.1186/1477-7525-2-43ResearchConstruction of a questionnaire measuring outpatients' opinion of quality of hospital consultation departments Gasquet Isabelle [email protected] Sylvie [email protected] Carla [email protected] Pierre [email protected] Philippe [email protected] Bruno [email protected] Delegation of innovation, health security and assessment, Direction of medical policy – Assistance Publique-Hôpitaux de Paris, 3 avenue Victoria 75184 Paris cedex, France2 UPRES JE 2360 (PSIGIM : Paris South Innovation Group in Mental health), Paul Brousse hospital, Villejuif, France3 Department of Public Health and Medical Informatics, Faculté de Médecine Broussais Hôtel Dieu, Paris, France4 Department of Epidemiology, Biostatistics et and clinical research, Hôpital Bichat Assistance Publique-Hôpitaux de Paris, Paris, France2004 4 8 2004 2 43 43 21 5 2004 4 8 2004 Copyright © 2004 Gasquet et al; licensee BioMed Central Ltd.2004Gasquet et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Few questionnaires on outpatients' satisfaction with hospital exist. All have been constructed without giving enough room for the patient's point of view in the validation procedure. The main objective was to develop, according to psychometric standards, a self-administered generic outpatient questionnaire exploring opinion on quality of hospital care. Method First, a qualitative phase was conducted to generate items and identify domains using critical analysis incident technique and literature review. A list of easily comprehensible non-redundant items was defined using Delphi technique and a pilot study on outpatients. This phase involved outpatients, patient association representatives and experts. The second step was a quantitative validation phase comprised a multicenter study in 3 hospitals, 10 departments and 1007 outpatients. It was designed to select items, identify dimensions, measure reliability, internal and concurrent validity. Patients were randomized according to the place of questionnaire completion (hospital v. home) (participation rate = 65%). Third, a mail-back study on 2 departments and 248 outpatients was conducted to replicate the validation (participation rate = 57%). Results A 27-item questionnaire comprising 4 subscales (appointment making, reception facilities, waiting time and consultation with the doctor). The factorial structure was satisfactory (loading >0.50 on each subscale for all items, except one item). Interscale correlations ranged from 0.42 to 0.59, Cronbach α coefficients ranged from 0.79 to 0.94. All Item-scale correlations were higher than 0.40. Test-retest intraclass coefficients ranged from 0.69 to 0.85. A unidimensional 9-item version was produced by selection of one third of the items within each subscale with the strongest loading on the principal component and the best item-scale correlation corrected for overlap. Factors related to satisfaction level independent from departments were age, previous consultations in the department and satisfaction with life. Completion at hospital immediately after consultation led to an overestimation of satisfaction. No satisfaction score differences existed between spontaneous respondents and patients responding after reminder(s). Conclusion Good estimation of patient opinion on hospital consultation performance was obtained with these questionnaires. When comparing performances between departments or the same department over time scores need to be adjusted on 3 variables that influence satisfaction independently from department. Completion of the questionnaire at home is preferable to completion in the consultation facility and reminders are not necessary to produce non-biased data. Patient satisfactionquality of carehospitalconsultationpsychometrics ==== Body Background Medical care aims not only to improve health status but also to respond to patient needs and wishes and to ensure their satisfaction with care [1]. Likewise, conducting surveys to measure satisfaction with psychometrically validated questionnaires entails assessment of the quality of care organization and procedures [2]. Patient judgement on medical care also contributes to medical outcome. In the case of ambulatory care, it has been clearly shown that satisfied patients are more likely to cooperate with treatment, to maintain a continuing relationship with a practitioner [3] and thus enjoy a better medical prognosis [4]. From a conceptual point of view, the construct of patient satisfaction as been defined by Ware as an "attempt to capture the personal evaluation of care that cannot be known by observing care directly" and to consider opinion of patients as a multidimensional subjective indicator of quality of care [5]. The model most commonly, though implicitly, used in satisfaction work is the discrepancy model (degree of fulfillment of expectation is related to satisfaction level) giving to patient expectations a central role [6]. This model, according to Sitzia " implies that concentrating upon areas of expressed dissatisfaction is more valuable than obtaining consistency of expressed satisfaction" [4,7]. In France, measuring satisfaction has been mandatory since 1996 and several questionnaires have been developed to evaluate inpatient care [8-12]. Most existing outpatient satisfaction questionnaires have been developed to assess primary care practice, especially general practice [13-20]. However, it could be hypothesized that content of questionnaires evaluating primary care physician may be different from that of questionnaires exploring hospital consultation with a specialist because of differences in patient expectations. So it could be assumed that dimensions that are very important in the case of primary care like human qualities of the physician and medical information could have a lesser importance in case of hospital consultation, while technical competency could have a more important place [21-23]. Few questionnaires have been developed for hospital consultations. Of these, some were specific to one type of consultation like oncology [24], rheumatology [25] or diabetes clinics [21], while others were non-generic questionnaires [14]. There is one French-language questionnaire on satisfaction with outpatient hospital care, however this questionnaire was developed from an "expert" viewpoint [26]. Hence the decision to construct a complementary "patient-oriented" questionnaire implicating potential respondents in the generation and selection of items. Even if health care organization differs across countries, the role of the hospital in most countries is very similar and it could be expected that the questionnaire developed in France could be used in other countries with a public health system, in particular European countries. The main objective was to develop, according to psychometric standards, a generic outpatient satisfaction questionnaire that could be used to compare hospital outpatient departments one with another or the same department over time. The questionnaire needed to be brief, understandable and easy to complete for outpatients aged18 years or older in medicine, surgery and psychiatric hospital consultations. It was designed to be self-administered. The French final version is being adapted in English, German, Italian, Spanish and Hebrew. The secondary objective was to define administration procedures in routine study that minimize non-response bias. Three situations were tested: i) questionnaire issued and completed at hospital, immediately after consultation; ii) questionnaire mailed and completed at home before any reminder; iii) questionnaire mailed and completed at home only after reminder(s). The groups were compared for satisfaction. Overview of the questionnaire development It comprised 2 phases. First, a qualitative phase for item generation and construction of a first version of the questionnaire (41-item version). Secondly, a quantitative phase comprising 2 steps. A first validation phase that provided a shortened version of the questionnaire (27-item version). Second, a replication validation phase to corroborate results from the previous steps. Finally a very short-form version (9-item) was constructed. All versions are presented in the Appendix (see additional file 1). A steering committee supervised the questionnaire development procedure, comprising methodologists, hospital practitioners and persons from patient associations defending health care user rights. All analyses were performed using SAS software (version 8). Method Qualitative phase of item generation A psychologist conducted 25 individual semi-structured interviews with recent outpatients, using the critical analysis incident technique [27]. Subjects were asked to detail specific events they had experienced and situations associated with neutral, pleasant or unpleasant emotions that had influenced their opinion on consultation. An interview guide constructed according to the chronological order of a consultation was used. The interviews were pursued until new ideas were exhausted. Patients expressing ideas that were too general or those talking about non-personal experiences were interrupted in order to refocus on a particular personal experience. Each interview lasted 30 minutes on average. All the different wordings of a given idea were recorded. Interviews were transcribed and items were generated from the verbatim statements (n = 105 items). A literature review was carried out on validated satisfaction questionnaires [5,13-20,23-26,28-30]. This yielded a preliminary list of areas of satisfaction with consultation. Items found in the literature but not in the interviews were collated (n = 26). This procedure also identified other factors related to outpatient satisfaction with consultation (patient and physician profiles), relevant for inclusion in the questionnaire. The aim was to select the variables linked to satisfaction, independent from place of consultation (department), for the final questionnaire. These variables constitute background adjustment factors needed to avoid bias in comparing departments one with another or the same department over time (age of the patient for example) [31]. A list of satisfaction items (n = 131) was constructed classified into the following domains: administrative procedures, appointment making, receptionist and nurses, waiting time, facilities, duration and privacy of the consultation with the doctor, human relationships with the doctor, information provided by the doctor and shared decision-making, doctor's technical competence, coordination and continuity of care, and global satisfaction. The source of items (interview v. literature) was indicated. Using the Delphi technique [32], the steering committee and six patients (members of the National League against Cancer) selected items within each domain (n = 60). The number of items to be chosen was proportional to the number of items proposed in each domain. The list of items was submitted as often as necessary to obtain a consensus of at least 80% among the raters. A focus group (one two-hour meeting) coordinated by two of the authors (IG, SV) including two patient association representatives and three patients, with previous individual access to the list of items, checked acceptability of item wording and exhaustiveness of the list. A pilot study was conducted on 55 outpatients from different outpatient departments using a preliminary questionnaire comprising the selected items, to check comprehensibility and acceptability of items and response patterns. Confusing items were removed, rewritten or replaced. The list of the items extracted from this qualitative phase is shown on the appendix. Questionnaire The questionnaire obtained from the qualitative phase and tested in the first study comprised 41 negatively and positively worded satisfaction items (Appendix [see Additional File 1]). The traditional approach was chosen, in which the item is structured as a statement of opinion. A Likert five-point response balanced scale was chosen (in French : 'yes certainly', 'yes probably', 'neither yes nor no', 'probably not, 'certainly not') because it seems to be the best format [5,33] and the most often used [5,13,14,17-19,24,28,29]. A 'does not apply' category was provided for 19 items relating to situations not universally relevant. Each item was scored from 0 to 4, 4 indicating greatest of satisfaction. Non-response and 'does not apply' categories were considered as missing data. Patients were asked to answer for their last consultation in the department. Several other items on general satisfaction were also included in this questionnaire: one overall satisfaction item, using a seven-point scale (from 'not at all' to 'completely' satisfied) and two items on intended behavior (to recommend, to consult again), using a four-point scale ('yes certainly', 'yes probably', 'probably not', 'certainly not') and one open-ended question. These items were included to test concurrent validity. The questionnaire also comprised data on sociodemographic profile, medical status, visit background and characteristics. and an overall satisfaction with life (using a 7 point scale, from 'not at all' to 'completely' satisfied). This last variable was included because of the relationship between affective disposition and the expression of satisfaction [34,35] and because of the relationship between satisfaction with life and satisfaction with care [36]. Samples and studies design of the quantitative phase First study (first validation phase) To select items, a first study was conducted in 2001–2002 in 10 wards of 3 short-stay public teaching hospitals of Paris area (Paul Brousse, Bichat and George Pompidou European hospitals). Data was collected in 7 medical departments (internal medicine, rheumatology, 2 cardiology, dermatology, infectious disease, and oncology) and 3 surgical outpatient departments (urology, orthopedic, surgical gynecology). All consecutive eligible ambulatory patients over 18 years in scheduled consultation with a physician were included, to obtain approximately 100 subjects per department. Patients hospitalized before or immediately after the consultation were excluded. Research assistants approached outpatients immediately after consultation and invited them to participate. Outpatients were randomized prior to being approached. Outpatients randomized in group 1 completed the questionnaire alone immediately after consultation and left it in a box. Patients of group 2 received the questionnaire by mail at home for completion. They were asked to complete and return it by post in a prepaid envelope carrying a neutral address. Non-respondents were sent up to 3 more questionnaires at one-week intervals. To assess reliability over time a sample of 38 respondents from the second group was sent a second questionnaire to return completed, without any reminder. Finally of the 1548 outpatients approached, 70.9% agreed to participate (n = 1097) and 65.1% completed the questionnaire (n = 1007). Response rates were 57.0% in group 1 and 73.7% in group 2 (40.2% before any reminder, 63 % after one reminder, 69.7% after two and 73.7% after three). Reasons for non-participation were refusal or lack of time (12.9% of the overall sample), language barrier (8.5%), inability for medical reasons (7.2%), other reason (0.6%) and agreement but no return of the questionnaire after 3 reminders (5.8% of the overall sample and 12.0% of group 2). Compared to respondents, the non-respondent group comprised older subjects (60.2% v. 52.6% aged over 50 years, p < 0.001), more foreigners (12.5% v. 29.1%, p < 0.001) and more patients consulting for the first time in the department (28.0% v. 22.4%, p = 0.02). Response rates also differed according to the department (p < 0.001) and the hospital (p < 0.001). Second study (replication phase) To confirm the results of the previous study, a second study was conducted in the year 2002 in two departments (internal medicine and infectious disease) in one short-stay public teaching hospital. All consecutive outpatients of 18 years and over (not hospitalized immediately after consultation) were included to obtain 100 participants per department. The questionnaires were posted with a prepaid envelope. One reminder was sent 10 days after the first mailing to non-respondents. Participation rate was 33.9% before reminder and 56.5% after (n = 248). Results First validation phase Item selection A first selection of items was made from descriptive response distribution for each item. The criteria used to guide item selection/deletion were: high rates of non-response and 'not applicable' response (≥ 20%) except for items where high rates in this response category were expected, ceiling and floor effects (≥ 50%), and unacceptable test-retest reliability (weighted kappa coefficient<0.60). Pragmatic considerations also tempered selection: interest of the item in itself, number of items covering the same domain, redundancy. Results showed that the proportion of missing responses per item was low. As predictable, for the two items relating to accessibility of the service in case of emergency (items 5 and 6, Appendix [see Additional File 1]) the number of 'does not apply' responses was high (30.7% and 45.0%). A ceiling effect was observed for all items (from 54.4% to 79.6%), except for those on facilities and waiting time (items 10 to 13). Test-retest reliability was good for 20 items (weighted kappa ≥ 0.7 for 10 items and from 0.6 to 0.69 for 10 items). For 5 items, the coefficient ranged from 0.45 to 0.56. The item on doctors' warnings on side effects of treatment (item 22) had a very low weighted kappa (k = 0.17). At this stage 12 items were discarded. Item 22 was retained for its clinical relevance (Table 1). Table 1 Item description and scaling properties of the questionnaires extracted from the validation phase (26 item version) and from the replication phase Intermediate questionnaire 26-items retained at the and of the first validation phase (first study, n = 1007) Final questionnaire 27-item questionnaire tested at the replication phase (second study, n = 248) Title of the scales Consultation with the doctor Appointment making Reception Waiting time & facilities Overall scale Consultation with the doctor Appoint-ment making Reception & facilities Waiting time Overall scale Items properties # of items in the scale 13 6 3 4 26 13 6 5 3 27 # of questionnaires with at least 1/2 of items completed (1) 996 931 1004 1001 1003 235 248 247 244 248 # of items with 'non response' ≥ 20% 0 0 0 0 0 0 0 0 0 0 # of item with 'does not apply' response ≥ 20% 0 2 0 0 2 1 0 0 0 1 # of items with ceiling effect ≥ 50% (≥ 60%) 13 (12) 6 (4) 3 (2) 0 (0) 22 (18) 12 (4) 1 (1) 2 (0) 0 (0) 15 (5) # of items floor effect ≥ 50% 0 0 0 0 0 0 0 0 0 range of Weighted kappa (# of items with kappa ≥ 0.60) 0.14–0.83 (10) 0.46–0.77 (3) 0.45–0.78 (1) 0.68–0.82 (4) 0.14–0.82 (18) - - - - Scaling properties Mean score (± sd) 85.1 (17.2) 83.2 (19.9) 88.0 (14.5) 69.6 (24.9) 82.7 (13.7) 84.1 (17.2) 80.6 (18.5) 75.3 (18.3) 61.3 (19.6) 78.9 (15.3) Ceiling / floor effect (%) 26.2 / 0.10 32.2 / 0.2 38.5 / 0.1 16.2 / 1.1 4.2 / 0.1 25.8 / 0.4 24.7 / 0.4 13.7 / 0.5 19.0 / 4.9 4.4 / 0.4 Skewness value /SE -3.00 -2.09 -3.5 -0.86 -2.67 -0.98 -0.83 -0.58 -0.20 -0.76 Range of interscale correlations 0.33–0.35 0.34–0.37 0.35–0.40 0.33–0.40 - 0.46–0.51 0.51–0.59 0.42–0.49 0.42–0.53 - # of items with own scale correlation ≥ 0.40 (3) 12 6 2 4 - 13 6 5 3 - # of items with own scale (3) correlation greater than with other scale 13 6 3 4 - 13 6 4 3 - Cronbach alpha coefficient 0.85 0.82 0.69 0.77 0.88 0.94 0.87 0.79 0.89 0.94 Intraclass coefficient [IC95%] 0.69 [0.49–0.83] 0.84 [0.71–0.91] 0.86 [0.75–0.92] 0.83 [0.71–0.91] 0.90 [0.81–0.94] - - - - (1) Including non response and 'does not apply' response' (2) From the final principal component factor analysis (3) Corrected for overlap Factorial structure The 29 items retained were entered into principal-components factor analysis (PCFA) with 'varimax' rotation and the 26 items with substantial loading ≥ 0.40) on only one factor were retained (Appendix [see Additional File 1]). Another PCFA was computed on the 26 remaining items to determine the structure of the instrument. The screeplot revealed a predominant eigenvalue with nevertheless a four-dimensional structure (the following eigenvalues showed a smooth decrease). Hence the proposal is to consider a four-dimension structure with the possibility of an overall score. One dimension grouped the 13 items relating to consultation with the physician. The second dimension grouped the 6 variables relating to appointment-making. The third and fourth related respectively to waiting time or facilities (4 items) and reception (3 items). None of the 26 items loaded on more than one factor. Only item 26 ('doctor in touch with attending physician') had a borderline loading (0.37), but it was kept because coordination of care in hospital care is important. The stability of the 4 factors was ascertained with PCFA on subgroups and with 'oblique' rotation (male v. female and surgery v. medicine). Scale properties Scores for each scale were based on the standardized sum of the items, giving a range from 0 (low satisfaction) to 100 (high satisfaction). Scores were computed when at least half the items in a scale were completed. Because of a ceiling effect, mean scale scores are relatively high except for the 'waiting time and facilities' scale (Table 1). Interscale correlations were good for the four scales. One item had a borderline correlation with its own scale (r = 0.37 for item 7 'the consultation room was clearly sign-posted') and one item had a low correlation (r = 0.33 for item 26 'doctor in touch with attending physician'). All items had a higher correlation to their hypothesized scales than to other scales. Reliability was good, meeting both Cronbach alpha and intraclass correlation coefficient requirements (Table 1). A very strong association between the overall scale, intended behaviors, comments and global satisfaction question was noted, suggesting good concurrent validity (Table 2). Table 2 Association between overall satisfaction scale, intended behaviors and global satisfaction item from the first validation study (n = 1007) and replication study (n = 248) First validation phase First study (n = 1007) Replication phase Second study (n = 248) n Score (sd) (1) P n Score (sd) (2) P Overall satisfaction item (3) 1 (not at all satisfied) 13 58.0 (21.6) 2 12 68.7 (17.7) 3 18 56.0 (13.5) <0.001 (4) - na - 4 39 59.4 (14.0) 5 125 72.1 (11.5) 6 285 80.3 (10.0) 7 (completely satisfied) 496 90.6 (7.6) To recommend to relatives or friends certainly not 13 52.7 (18.2) <0.001 (4) - na - probably not 30 57.3 (16.7) yes probably 272 75.4 (13.1) yes certainly 665 87.6 (9.5) To consult again certainly not 10 58.7 (24.2) <0.001 (4) - na - probably not 20 57.3 (17.9) yes probably 213 72.5 (14.5) yes certainly 756 86.6 (10.3) To consult again 9 Do not agree - na - 46 57.8 (14.4) <0.001 agree 18 64.2 (12.4) Fully agree 1 84.3 (11.7 Content of the open-ended question negative comment 303 76.1 (14.7) 85 72.1 (14.9) mixed comment 110 78.4 (13.5) <0.001 8 81.0 (12.4) <0.001 no comment 442 85.4 (12.1) 91 82.0 (15.8) positive comment 152 91.2 (8.2) 42 85.5 (11.1) na: non available (1) Overall satisfaction score (26 items scale extracted from the first study) (2) Overall satisfaction score (27 items final scale extracted from the second study) (3) 7-point scale from 1 'not at all satisfied' to 7 'completely satisfied' (4) ANOVA test regrouping the responses 1,2 and 3 Replication phase Questionnaire tested (see appendix) A modified version of the questionnaire was constructed at the end of the previous step. To avoid the ceiling effect highlighted in the previous stage, responses choices were modified (using the pattern 'fully agree', 'agree', 'moderately agree, 'not really agree', 'not agree at all'). One satisfaction item on waiting time was added and one item on the facility was reworded to improve the chance of revealing a 'waiting time' subscale and a 'reception-facilities' subscale) and because reliability of the 'reception' subscale was borderline. Patient demographic variables identified at the previous stage as having a relationship with satisfaction scores, one item on intended behavior and an open-ended comment field were also added to the questionnaire. Final psychometric properties of the final 27-item version questionnaire The number of items with ceiling effect decreased. Item completion rates were good (Table 1). PCFA was performed on the 27 items. The screeplot highlighted the same internal structure. The 'varimax' rotation revealed that two dimensions were identical to those identified in the first study ('consultation with the doctor' and 'contact-appointment') (Table 4). The two others were slightly altered: the three items on 'waiting time' were isolated from items about 'facilities' that grouped themselves with the 'reception' factor. All items had a good loading on their own factor. Item 9 ('pleasantness and availability of receptionist') was the only item with secondary loading on another component. It was kept because it was the only item on human qualities of non medical staff which were cited very often by patients in the qualitative phase (Table 1). Table 4 Principal components factor analysis (varimax rotation) computed with the final 27-items version of the questionnaire (second study, n = 248) Factor 1 Factor 2 Factor 3 Factor 4 Consultation with the doctor Appointment making Reception & facilities Waiting time 1 easy to make an appointment by phone 0.07 0.49 0.12 0.39 2 Pleasantness of staff answering the phone 0.24 0.56 0.30 0.19 3 Acceptable time lapse to obtain appointment 0.19 0.81 0.22 0.18 4 Possibility of obtaining an appointment on convenient day and hour 0.20 0.77 0.20 0.14 5 Contacting someone in the facility on the phone for help or advice in case of problem 0.30 0.68 0.18 0.08 6 In an emergency, getting a quick appointment in the facility 0.19 0.77 0.24 0.11 7 Inside the hospital the consultation room was clearly sign-posted' 0.17 0.15 0.67 - 0.16 8 Administrative procedures (completing papers and paying) fast and easy 0.15 0.25 0.61 0.16 9 Pleasantness and availability of receptionist 0.20 0.44 0.59 0.23 10 Waiting room pleasant 0.11 0.15 0.78 0.26 11 Premises clean 0.17 0.22 0.66 0.18 12 Saw the doctor at the appointed time 0.24 0.18 0.14 0.84 13 Waiting time acceptable 0.25 0.29 0.10 0.85 14 Information on how long to plan for 0.23 0.16 0.20 0.78 15 The doctor was welcoming 0.69 0.09 0.38 0.08 16 Took an interest in me not just my medical problem 0.72 0.06 0.15 0.18 17 spent adequate time with me 0.82 0.14 0.21 0.07 18 Examined me carefully 0.78 0.04 0.21 0.07 19 Explained what he/she was doing during the consultation 0.78 0.15 0.17 0.12 20 Wanted to know if I had pain 0.75 0.13 0.17 0.15 21 Asked if I was taking medication for other health problems 0.72 0.07 0.08 0.09 22 Warned me about possible side effects of treatment (operation, drugs)- 0.68 0.30 0.01 0.19 23 Took my opinion into account 0.79 0.20 0.02 0.10 24 Explained decisions 0.76 0.25 0.18 0.20 25 I got the information I wanted 0.78 0.23 0.12 0.17 26 he/she is in touch with my GP 0.57 0.20 0.02 0.15 27 Agree with doctor's instruction 0.49 0.32 0.04 - 0.06 For item-scale correlations, item 9 also correlated to these two scales ('reception-facilities' factor and 'contact-appointment'). It was decided to attribute it to the factor that maximized internal consistency ('reception-facilities' scale). All items met the requirement of being highly correlated to their own scale, all interscale correlations were satisfying, as well as internal consistency (Table 1). The scale overall was significantly associated with comments and intended behaviors (Table 2). Construction of a unidimensional 9-item form As the factorial analysis of both the first validation and replication phases revealed a predominant factor that could be split into four underlying dimensions, it was decided to construct a unidimensional form of the questionnaire, that could produce an overall global outcome that could be very useful in case of evaluation study. Within each dimension, one third of the items were selected according to two criteria: items without 'not applicable' response choice, and items having strong loading on the principal component in PCFA. Thus 9 items were selected, 4 items from the 'consultation with the doctor' scale, 2 from the 'contact-appointment' scale, 2 from 'reception-facilities' and 1 from 'waiting time' (Appendix [see Additional File 1]). Final PCFA on these 9 items showed scale unidimensionality. Item loading on this factor ranged from 0.56 to 0.78. Item-scale correlation corrected for overlap ranged from 0.47 to 0.65. Internal consistency was good (Cronbach α = 0.86). Effect of mode of questionnaire administration on estimation of patient satisfaction First study showed that compared to the satisfaction score obtained with completion at home, mean scores for all hospital-completed satisfaction scales were very significantly higher. In the group that completed the questionnaire at home, comparison between respondents before any reminder and respondents after reminder(s) showed no difference in satisfaction scores, whatever the scale considered (Figure 1 – Satisfaction scores according to the place of completion and time of answering [before v. after reminder]). Figure 1 Satisfaction scores according to the place of completion and time of answering (before v. after reminder) (first study, n = 1007). Differences between departments A multiple linear regression showed that differences between departments were highly significant, even if patient characteristics that influenced patients' satisfaction were taken into account (i.e. age, satisfaction with life and previous consultation). Satisfaction scores ranged from 79.3 to 91.7 for 'consultation with the doctor' scale, from 72.8 to 94.2 for 'appointment making' scale, from 83.4 to 91.3 for 'reception' scale, from 57.3 to 80.5 for 'waiting time-facilities' and from 77.8 to 89.3 for the overall scale). Older age, good satisfaction with life and numerous previous consultations in the department were all associated with high levels of satisfaction, independently from the department (Table 3). Table 3 Association between demographic, medical, outpatient consultation characteristics considered as explanatory variables and overall satisfaction score as dependant variable (1) (linear regression analysis from the first study) DF F-value P-value Demographic profile Age (quantitative variable) 1 7.75 0.006 Matrimonial status (Married or living with partner v. single v. divorced, separated, or widowed) 2 0.93 0.42 Working status (employed v. student v. unemployed v. Retired v. prolonged sick leave v. other) 5 0.79 0.56 Level of education (university yes v. no) 1 1.72 0.19 Overall satisfaction with life (quantitative variable) 1 51.1 0.001 Modes of care provision Outpatient department (n = 10) 9 4.3 0.001 # of consultations in the department (first v. 2 to 3 v. 4 to 5 v. more then 5) 3 2.92 0.03 At least one hospitalization in the ward 1 0.73 0.39 Medical profile Duration of the health problem justifying the consultation (less than 6 month v. 6 month and more) 1 FGF 0.22 Severe medical problem ('yes definitely' v. 'yes rather' v. 'neither yes nor no', v. 'not really', v. 'definitely not' v. 'do not know') 5 1.19 0.31 Comorbidity (yes v. no) 1 1.97 0.16 Perceived health status, compared to persons of same age (better v. similar v. worse) 2 0.49 0.61 829 observations used in the analysis, r2 = 0.21 (1) First short version of the satisfaction questionnaire List of other variables not entered into the model because of non significance in the bivariate analysis (p > 0.1): gender, nationality, gender of the physician, motive of the consultation (physical, psychological or mixed) and prescription of test or medication and having a general practitioner. Discussion Psychometric properties of the scale The 27-item and 9-item versions of the questionnaire developed here appear sufficiently concise, valid and reliable to provide a non-biased subjective evaluation of outpatient viewpoint on the quality of care and services in hospital consultations. The questionnaire demonstrated very good internal consistency and good reliability over time. The construction strategy presented here follows most of the recommendations for "good practice" in validation of measurement tools of patient satisfaction with care [7]. Questionnaire content comprises culture-specific features, but overall remains consistent with various north American and European studies [21,23,26,37]. The predominant role given to patients in the early development stages, the literature review and the implication of various experts ensure good content, construct and face validity. This first qualitative step, often insufficiently detailed and structured in satisfaction questionnaire construction, is indeed crucial [38]. The quantitative phase (i.e. first validation with replication) used not only statistical and psychometric results to reach decisions, but also the "intrinsic" and "clinical" relevance of items. This is a very important point. First, because satisfaction studies aim not only to measure quality from user viewpoint, but also to highlight practical elements that can be modified to improve quality. Second, questionnaires that are perceived to have content validity are needed to generate interest in results among health professionals and provide incentive for changes in approach to their jobs. Third, the tendency of health professionals to develop "home-made" questionnaires and their reluctance to use validated questionnaires developed elsewhere can be countered if questionnaire items are perceived as relevant. Dimensions of the questionnaire Each dimension comprises items exploring both technical aspects of care (i.e. equipment, competence, accessibility, continuity, compliance, pain management, waiting and consultation time...) and interpersonal aspects of care (i.e. information, decision sharing, attitude...). These aspects are both predictors of patient opinion on care and services [22,23,37] because implementation of appropriate technical medical strategies is necessary, but not sufficient, to achieve desired outcome. Good management of the human is needed because, as Donabedian remarks, "the interpersonal process is the vehicle by which technical care is implemented and on which its success depends" [1]. According to this author, technical and interpersonal performances are the first circle around the "bull's eye" of the "quality of care" target. The most important dimension explaining outpatient opinion of hospital quality is the actual consultation with the physician, representing half the items in the tool. This is consistent with other generic patient questionnaires on satisfaction with ambulatory care, also comprising a majority of items related to the medical intervention [17,19,21,26,29,37]. No independent subscales regarding specific aspects of the patient-physician encounter (i.e. communication, professional competence, interpersonal skills...) were identified here. They have been regularly identified by authors developing GP satisfaction questionnaires [5,17,29,37,39,40]. This could be explained by the fact that, as hypothesized, expectations of outpatients with respect to hospital care differs from expectations from primary care. Possibly patients have different needs and expectations according to the type of consultation, hospital specialists generating more mixed expectations because the specific technical competence of hospital specialists predominates and patients have greater difficulty in dichotomizing doctors' skills into "affective " and "technical' dimensions [23,41,42], whereas "affective" qualities have a predominant role in primary care [15,16,43,44]. This is corroborated by the fact that generic questionnaires designed to evaluate hospital care (inpatient or outpatient) most often do not identify such human versus technical dimensions [8,10,26,45,46]. The three other dimensions ('contact-appointments', 'reception-facilities' and 'waiting time') are all related to organizational non-medical aspects of care. These dimensions are classically identified in other generic questionnaires [17,18,21,23,28,29,40,42]. Comparison of the two factorial structures shows stability for all dimensions except 'reception-facilities' and 'waiting time'. From a strictly psychometric viewpoint, these two dimensions, both exploring events occurring just care quality, these two dimensions can pinpoint independent improvement measures, and calculating two different scores may improve the probability of highlighting the impact of such measures. Differences between departments and role of background factors It was shown that satisfaction scores were strongly related to consultation department, regardless of outpatient, physician and care-provision characteristics. These results suggest that this measure is more sensitive to levels of department performance than to patient profile or to modes of consultation, as shown elsewhere [47]. Therefore it is important that each department should identify its weak points to implement specific targeted actions to improve care quality. As in numerous studies, it was observed that older patients have a higher opinion of care provided than others [7,23,48]. For several authors, this contributes to construct validity of satisfaction questionnaires [41]. The same was observed for patients with multiple contacts with a department [10]. This could be explained by a better match between expectations and experience for multiple consultants, dissatisfaction during first contact leading patients to consult elsewhere. The strong relationship between overall satisfaction with life and opinion on care expresses the influence of the individual affective disposition trait (i.e. general tendency of an individual to be optimistic or pessimistic) which influences job satisfaction, a concept very close to patient satisfaction with care [49]. Other studies have found relationships between satisfaction and variables strongly associated with perception of overall quality of life, like mental health status and health-related quality of life [12,24]. The influence of these three background factors suggests the need to adjust patient satisfaction scores on these three variables (i.e. patient age, number of contacts and satisfaction with life) when comparing performances between departments or measuring performance over time within departments [31]. Impact of data collection method For patients completing the questionnaire immediately after consultation in the hospital, satisfaction estimates were higher than in case of home completion, in spite of procedures to preserve anonymity and confidentiality at hospital. Little data exists on the impact of place of completion for self-administered questionnaires on satisfaction with consultation: two studies conclude that patients express less satisfaction when the questionnaire is completed at home rather than in the medical facility [13,50] and one concluded that there was no difference according to data collection methods, but lacked power because of small sample size [51]. This could be interpreted as an over-estimation of patient satisfaction in case of completion in the facility, patients being more prone to express their real opinion when they have more time to consider the consultation and are safely back home [13]. Moreover response rates in the hospital completion group were relatively low (57%) expressing both refusal to participate or inability to respond, and reluctance to answer a satisfaction questionnaire immediately after consultation because of long waiting time beforehand, or because a relative, an ambulance or a taxi is waiting to take the patient home. It could be concluded that completion at home may be better than immediately after consultation. In the present study, no difference was observed between respondents without reminder and respondents only after reminder(s). This result is in agreement with other studies assessing inpatient satisfaction [9,52]. It could be concluded that reminders are not necessary to produce non-biased data. Limitations This work entails several limitations. First, overall response rates only reached 65% despite reminders sent to patients receiving mailed questionnaires at home. However, unlike other studies, the response rates calculated did not exclude patients unable to respond for medical reason (i.e. who were very ill or did not understand French) and homeless patients giving an invalid address (shelter...). Second, non-respondents differed from respondents regarding two background factors influencing satisfaction levels, with over-sampling of less-satisfied subjects in the respondent group (young patients and first consultants). There are also differences in participation rates between departments that could lead to over-estimating real differences between departments, because the more satisfied outpatients within each department may have been excluded. Third, validation is a continuous process and further studies are required to confirm these first results. The experimental nature of these studies may have induced bias in questionnaire responses. So there is a need to replicate findings using confirmatory statistical methods (IRT or structural equation model for example) using the data from non experimental, routine studies. Conclusion Good estimation of patient opinion on hospital consultation can be obtained with these two questionnaires. When comparing performances between departments or the same department over time scores need to be adjusted on the three variables that influence satisfaction independently from department (patient age, previous consultation in the department and overall satisfaction with life score). Mail-back completion at home of the questionnaire seemed preferable to completion in the consultation facility immediately after the consultation. Reminders are not necessary to produce non-biased data. Authors' contribution IG – initiation of the research, supervision of the project and drafting the manuscript; SV – coordination of the 2 studies, participation in the interpretation of the results and revision of the draft paper; CDS – performing statistical analyses; PD and PR- participation in the conception, design and coordination of the research; BF – participation in the interpretation of the results, supervision of the statistical analysis and revision of the draft paper. Acknowledgments The authors would like to thank the members of the steering committee (Prof. P. Auquier, Mr JP Escande, Mr C Gilioli, Mr T Greacen, Prof. F Guillemin, Dr J Labarère, Prof. J Lellouch, Dr A Leplège, Dr C Pourin), the National League against Cancer, the participating departments of Paul Brousse hospital (Prof. C Jasmin, Prof. D Vittecoq), Georges Pompidou hospital (Prof. B Augereau, Prof. JN Fiessinger, Prof. JP Lemerle, Prof. PF Plouin) and Bichat-Claude Bernard hospital (Prof. B Crickx, Prof. L Boccon-Gibod, Prof. C Lejeunne, Prof. O Meyer, Prof. R Taurelle, Prof. A Vahanian). This study was funded by a 2000 national PHRC grant (Programme Hospitalier de Recherche Clinique) from the French Health Ministry. Approval was given by the CNIL (Commission national Informatique et Libertés). ==== Refs Donabedian A The quality of care. How can it be assessed? Jama 1988 260 1743 1748 3045356 10.1001/jama.260.12.1743 Cleary PD Edgman-Levitan S Health care quality. Incorporating consumer perspectives Jama 1997 278 1608 1612 9370508 10.1001/jama.278.19.1608 Deyo RA Inui TS Dropouts and broken appointments. A literature review and agenda for future research Med Care 1980 18 1146 1157 7432014 Williams B Patient satisfaction: a valid concept? Soc Sci Med 1994 38 509 516 8184314 10.1016/0277-9536(94)90247-X Ware J. E., Jr. Snyder MK Wright WR Davies AR Defining and measuring patient satisfaction with medical care Eval Program Plann 1983 6 247 263 10267253 10.1016/0149-7189(83)90005-8 Asadi-Lari M Tamburini M Gray D Patients' needs, satisfaction, and health related quality of life: Towards a comprehensive model Health Qual Life Outcomes 2004 2 32 15225377 10.1186/1477-7525-2-32 Sitzia J Wood N Patient satisfaction: a review of issues and concepts Soc Sci Med 1997 45 1829 1843 9447632 10.1016/S0277-9536(97)00128-7 Labarere J Francois P Auquier P Robert C Fourny M Development of a French inpatient satisfaction questionnaire Int J Qual Health Care 2001 13 99 108 11430670 10.1093/intqhc/13.2.99 Gasquet I Falissard B Ravaud P Impact of reminders and method of questionnaire distribution on patient response to mail-back satisfaction survey J Clin Epidemiol 2001 54 1174 1180 11675170 10.1016/S0895-4356(01)00387-0 Salomon L Gasquet I Mesbah M Ravaud P Construction of a scale measuring inpatients' opinion on quality of care Int J Qual Health Care 1999 11 507 516 10680947 10.1093/intqhc/11.6.507 Gasquet I Dehe S Gaudebout P Falissard B Regular visitors are not good substitutes for assessment of elderly patient satisfaction with nursing home care and services J Gerontol A Biol Sci Med Sci 2003 58 1036 1041 14630886 Thi PL Briancon S Empereur F Guillemin F Factors determining inpatient satisfaction with care Soc Sci Med 2002 54 493 504 11848270 10.1016/S0277-9536(01)00045-4 Kinnersley P Stott N Peters T Harvey I Hackett P A comparison of methods for measuring patient satisfaction with consultations in primary care Fam Pract 1996 13 41 51 8671103 Stump TE Dexter PR Tierney WM Wolinsky FD Measuring patient satisfaction with physicians among older and diseased adults in a primary care municipal outpatient setting. An examination of three instruments Med Care 1995 33 958 972 7666709 Hjortdahl P Laerum E Continuity of care in general practice: effect on patient satisfaction Bmj 1992 304 1287 1290 1606434 Hopton JL Howie JG Porter AM The need for another look at the patient in general practice satisfaction surveys Fam Pract 1993 10 82 87 8477901 Baker R Development of a questionnaire to assess patients' satisfaction with consultations in general practice Br J Gen Pract 1990 40 487 490 2282225 McKinley RK Manku-Scott T Hastings AM French DP Baker R Reliability and validity of a new measure of patient satisfaction with out of hours primary medical care in the United Kingdom: development of a patient questionnaire Bmj 1997 314 193 198 9022436 Grogan S Conner M Willits D Norman P Development of a questionnaire to measure patients' satisfaction with general practitioners' services Br J Gen Pract 1995 45 525 529 7492421 Howie JG Heaney DJ Maxwell M Walker JJ A comparison of a Patient Enablement Instrument (PEI) against two established satisfaction scales as an outcome measure of primary care consultations Fam Pract 1998 15 165 171 9613486 10.1093/fampra/15.2.165 Westaway MS Rheeder P Van Zyl DG Seager JR Interpersonal and organizational dimensions of patient satisfaction: the moderating effects of health status Int J Qual Health Care 2003 15 337 344 12930049 10.1093/intqhc/mzg042 Cheng SH Yang MC Chiang TL Patient satisfaction with and recommendation of a hospital: effects of interpersonal and technical aspects of hospital care Int J Qual Health Care 2003 15 345 355 12930050 10.1093/intqhc/mzg045 Williams SJ Calnan M Convergence and divergence: assessing criteria of consumer satisfaction across general practice, dental and hospital care settings Soc Sci Med 1991 33 707 716 1957191 10.1016/0277-9536(91)90025-8 Loblaw DA Bezjak A Bunston T Development and testing of a visit-specific patient satisfaction questionnaire: the Princess Margaret Hospital Satisfaction With Doctor Questionnaire J Clin Oncol 1999 17 1931 1938 10561235 van Campen C Sixma HJ Kerssens JJ Peters L Rasker JJ Assessing patients' priorities and perceptions of the quality of health care: the development of the QUOTE-Rheumatic-Patients instrument Br J Rheumatol 1998 37 362 368 9619883 10.1093/rheumatology/37.4.362 Labarere J Francois P Bertrand D Peyrin JC Robert C Fourny M Outpatient satisfaction: validation of a French-language questionnaire: data quality and identification of associated factors Clin Perform Qual Health Care 1999 7 63 69 10747567 Norman IJ Redfern SJ Tomalin DA Oliver S Developing Flanagan's critical incident technique to elicit indicators of high and low quality nursing care from patients and their nurses J Adv Nurs 1992 17 590 600 1602075 Woodward CA Ostbye T Craighead J Gold G Wenghofer EF Patient satisfaction as an indicator of quality care in independent health facilities: developing and assessing a tool to enhance public accountability Am J Med Qual 2000 15 94 105 10872259 Rubin HR Gandek B Rogers WH Kosinski M McHorney CA Ware J. E., Jr. Patients' ratings of outpatient visits in different practice settings. Results from the Medical Outcomes Study Jama 1993 270 835 840 8340982 10.1001/jama.270.7.835 Sixma HJ Kerssens JJ Campen CV Peters L Quality of care from the patients' perspective: from theoretical concept to a new measuring instrument Health Expect 1998 1 82 95 11281863 10.1046/j.1369-6513.1998.00004.x Rahmqvist M Patient satisfaction in relation to age, health status and other background factors: a model for comparisons of care units Int J Qual Health Care 2001 13 385 390 11669566 10.1093/intqhc/13.5.385 Whitman NI The Delphi technique as an alternative for committee meetings J Nurs Educ 1990 29 377 379 2175780 Hendriks AA Vrielink MR Smets EM van Es SQ De Haes JC Improving the assessment of (in)patients' satisfaction with hospital care Med Care 2001 39 270 283 11242321 10.1097/00005650-200103000-00007 Duffy MK Ganster DC Shaw JD Positive affectivity and negative outcomes: the role of tenure and job satisfaction J Appl Psychol 1998 83 950 959 9885200 10.1037//0021-9010.83.6.950 Chang EC Sanna LJ Optimism, pessimism, and positive and negative affectivity in middle-aged adults: a test of a cognitive-affective model of psychological adjustment Psychol Aging 2001 16 524 531 11554528 10.1037//0882-7974.16.3.524 Bredart A Razavi D Robertson C Batel-Copel L Larsson G Lichosik D Meyza J Schraub S von Essen L de Haes JC A comprehensive assessment of satisfaction with care: preliminary psychometric analysis in French, Polish, Swedish and Italian oncology patients Patient Educ Couns 2001 43 243 252 11384822 10.1016/S0738-3991(00)00165-8 Williams SJ Calnan M Key determinants of consumer satisfaction with general practice Fam Pract 1991 8 237 242 1959723 Sitzia J How valid and reliable are patient satisfaction data? An analysis of 195 studies Int J Qual Health Care 1999 11 319 328 10501602 10.1093/intqhc/11.4.319 Haddad S Potvin L Roberge D Pineault R Remondin M Patient perception of quality following a visit to a doctor in a primary care unit Fam Pract 2000 17 21 29 10673484 10.1093/fampra/17.1.21 Poulton BC Use of the consultation satisfaction questionnaire to examine patients' satisfaction with general practitioners and community nurses: reliability, replicability and discriminant validity Br J Gen Pract 1996 46 26 31 8745848 Grogan S Conner M Norman P Willits D Porter I Validation of a questionnaire measuring patient satisfaction with general practitioner services Qual Health Care 2000 9 210 215 11101705 10.1136/qhc.9.4.210 Lewis JR Patient views on quality care in general practice: literature review Soc Sci Med 1994 39 655 670 7973865 10.1016/0277-9536(94)90022-1 Brody DS Miller SM Lerman CE Smith DG Lazaro CG Blum MJ The relationship between patients' satisfaction with their physicians and perceptions about interventions they desired and received Med Care 1989 27 1027 1035 2586185 Sixma HJ Spreeuwenberg PM van der Pasch MA Patient satisfaction with the general practitioner: a two-level analysis Med Care 1998 36 212 229 9475475 10.1097/00005650-199802000-00010 Carey RG Seibert JH A patient survey system to measure quality improvement: questionnaire reliability and validity Med Care 1993 31 834 845 8366685 Delbanco TL Enriching the doctor-patient relationship by inviting the patient's perspective Ann Intern Med 1992 116 414 418 1736775 Winefield HR Murrell TG Clifford J Process and outcomes in general practice consultations: problems in defining high quality care Soc Sci Med 1995 41 969 975 8545671 10.1016/0277-9536(94)00403-G Hall JA Dornan MC Patient sociodemographic characteristics as predictors of satisfaction with medical care: a meta-analysis Soc Sci Med 1990 30 811 818 2138357 10.1016/0277-9536(90)90205-7 Judge TA Bretz JR Report on an alternative measure of affective disposition. Educational and Psychological Measurement 1993 53 1095 1104 Savage R Armstrong D Effect of a general practitioner's consulting style on patients' satisfaction: a controlled study Bmj 1990 301 968 970 2282112 Trandel-Korenchuk DM Comparison of three visit-specific patient satisfaction instruments: reliability and validity measures and the effect of four methods of data collection on dimensions of patient satisfaction J Ambul Care Manage 1997 20 56 73 10181624 Lasek RJ Barkley W Harper DL Rosenthal GE An evaluation of the impact of nonresponse bias on patient satisfaction surveys Med Care 1997 35 646 652 9191708 10.1097/00005650-199706000-00009

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Health Qual Life Outcomes

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==== Front BMC MedBMC Medicine1741-7015BioMed Central London 1741-7015-2-291531765010.1186/1741-7015-2-29Research ArticleStress, burnout and doctors' attitudes to work are determined by personality and learning style: A twelve year longitudinal study of UK medical graduates McManus IC [email protected] A [email protected] E [email protected] Department of Psychology, University College London, Gower Street, London WC1E 6BT, United Kingdom2 London Department of Postgraduate Medical and Dental Education, 22 Guilford Street, London WC1N 1DZ, United Kingdom2004 18 8 2004 2 29 29 27 3 2004 18 8 2004 Copyright © 2004 McManus et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The study investigated the extent to which approaches to work, workplace climate, stress, burnout and satisfaction with medicine as a career in doctors aged about thirty are predicted by measures of learning style and personality measured five to twelve years earlier when the doctors were applicants to medical school or were medical students. Methods Prospective study of a large cohort of doctors. The participants were first studied when they applied to any of five UK medical schools in 1990. Postal questionnaires were sent to all doctors with a traceable address on the current or a previous Medical Register. The current questionnaire included measures of Approaches to Work, Workplace Climate, stress (General Health Questionnaire), burnout (Maslach Burnout Inventory), and satisfaction with medicine as a career and personality (Big Five). Previous questionnaires had included measures of learning style (Study Process Questionnaire) and personality. Results Doctors' approaches to work were predicted by study habits and learning styles, both at application to medical school and in the final year. How doctors perceive their workplace climate and workload is predicted both by approaches to work and by measures of stress, burnout and satisfaction with medicine. These characteristics are partially predicted by trait measures of personality taken five years earlier. Stress, burnout and satisfaction also correlate with trait measures of personality taken five years earlier. Conclusions Differences in approach to work and perceived workplace climate seem mainly to reflect stable, long-term individual differences in doctors themselves, reflected in measures of personality and learning style. ==== Body Background Sir William Osler (1849–1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives: "To each one of you the practice of medicine will be very much as you make it – to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man."[1] The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor's work changes, in part due to career development and progression through different jobs. In an important study, Delva et al [2] used earlier research [3,4] to develop two separate instruments for studying how doctors work, the Approach to Work Questionnaire (AWQ) and the Workplace Climate Questionnaire (WCQ). In Canadian physicians [2,5] the AWQ showed three separate factors, which were called Surface-Rational, Surface-Disorganised, and Deep (see table 1). These approaches related to different methods and motivations for continuing medical education. Those with a deep approach preferred independent and problem-based learning and motivation was internal. Surface-rational and surface-disorganised approaches were primarily driven by external motivation, with the preferred mode of continuing education learning being independent for the surface-rational, and in consultations for the surface-disorganised. The WCQ showed three dimensions, called Choice-Independence, Supportive-Receptive, and Workload (see table 1), which correlated with the AWQ. Doctors reporting Choice-Independence and Supportive-Receptive work environments had a Deeper approach, whereas those describing an environment dominated by Workload tended to be more Surface-Disorganised. Some doctors are unhappy with their work, which can manifest as stress (usually assessed by the General Health Questionnaire) or burnout, which has three separate components of emotional exhaustion, depersonalisation and reduced personal accomplishment (see table 2). Greater stress and burnout in doctors are related to the personality trait of neuroticism or 'negative affectivity' [6]. The AWQ and WCQ provide a snapshot of a doctor's learning environment and approach to work at one particular time, as also do measures of stress and burnout. A key question, as Deary et al recognised [6] when considering stress, is the extent to which different approaches to work and the climate of the workplace are consequences of the workplace or of the doctor. At first sight it might seem that the workplace itself has to be the primary force driving both workplace learning and workplace climate. However, it is also possible that approaches to learning and work mainly depend upon pre-existing differences among doctors, differences that may already have manifested earlier in the doctors' careers. The AWQ bears a strong formal similarity to the surface, deep and strategic study habits and learning styles identified by the Study Process Questionnaire (SPQ), which assesses the motivations and approaches used by students in higher education (see table 3). The similarity is not accidental since the AWQ was developed by adapting items from Entwistle and Ramsden's Approaches to Study Inventory [7], which has a similar factor structure to that of the Study Process Questionnaire [8]. It is therefore expected that there may be significant continuities across approaches to study and approaches to work. In this paper we describe a large cohort of UK doctors, typically aged 29 or 30 at the time of the study, who have been qualified for five or six years, who are practising as SHOs or SpRs in hospital or are in general practice, and who previously had been studied when aged 17 or 18 at application to medical school in autumn 1990 [9], in their final year at medical school [10] and as PRHOs [11]. The main interest here will be in the extent to which a doctor's present approaches to work and their workplace climate, as well as their stress and burnout, relate to earlier measures of study habits and personality at application to medical school and subsequently. Method Participants In the autumn of 1990 a questionnaire was sent to all individuals with European Community postal addresses who had applied to any of the five UK medical schools taking part in the study [9]; they represented about 70% of all applicants and acceptances for medical school in that year. The response rate was 93%. Students who were accepted for entry in 1991, 1992 or 1993 were followed up in their final year at medical school (1995–1998), when the response rate was 56%, and at the end of their PRHO year (1996–1999), when the response rate was 58%. In 2002 a tracing exercise searched the Medical Register and Medical Directory from 1995 to 2002 to find the addresses of as many doctors as possible who were in the original survey, and who were known not to have died, left medical school during basic medical sciences, or otherwise to be no longer in the survey. For study design see figure 1. Questionnaire Questionnaires were sent to all individuals with current or recent GMC addresses. The questionnaire consisted of a single folded A3 sheet of paper (4 A4 sides). Included in the present questionnaire (described in the results as '2002') were the 12-item General Health Questionnaire (GHQ) [12]; an abbreviated version of the Maslach Burnout Inventory (aMBI), which has three sub-scales, Emotional Exhaustion, Depersonalisation, and Personal Accomplishment [13,14]; a three-item scale modelled on the aMBI, which assesses Happiness with a Medical Career [15]; an abbreviated version of the Study Process Questionnaire, which has three sub-scales of Surface, Strategic and Deep learning [16,17]; an abbreviated questionnaire assessing the 'Big Five' personality dimensions of Neuroticism, Extraversion, Openness to Experience, Agreeableness and Conscientiousness [15,18]; and abbreviated versions of the Approach to Work Questionnaire (aAWQ) and the Workplace Climate Questionnaire (aWCQ) [19], each of which has three sub-scales, and for which a detailed description is provided in the Supplementary Information (see Additional file: 1) [2]. The GHQ, aMBI and personality questionnaire had also been administered previously in the PRHO survey, and the SPQ had been administered in the Applicant and Final year surveys. Procedure Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25th March 2003, a few questionnaires were returned up until the end of August 2003. Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52. Results The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias (see Supplementary Information see Additional file: 1). Respondents The mean age of respondents on 1st December 2002 was 30.4 years (SD 1.86, range 28.3 – 49.2). There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere [19] (see Supplementary Information see Additional file: 1). There was also substantial variation on the measures of stress, burnout and satisfaction with medicine as a career, with 21.3% of doctors (345/1617) reporting GHQ scores of 4 or more, the conventional level of 'caseness'. Approaches to work and learning were correlated with climate in the workplace, and as in the Delva et al study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table 4). Approaches to work Table 4 shows correlation of the stress measures with approaches to work and study habits. The largest correlations were of a surface-rational approach with a strategic learning style, and a deep approach to work with a deep learning style. In each case the correlations were not only highly significant when study habits were measured in the final year at medical school, six or seven years earlier, but were also very significantly correlated with study habits measured at selection, twelve years earlier. Correlations of approaches to work and stress, burnout and satisfaction with medicine were generally small, and generally were only with measures taken in 2002, and not with measures taken as a PRHO, five or six years earlier. The sole exception was that a surface-disorganised approach correlated with high stress as measured by the GHQ, both in 2002 and with stress when the doctors were PRHOs. Workplace climate Table 5 shows correlations between the workplace climate and study habits, stress, burnout and satisfaction with medicine. In contrast to the associations with approaches to work, the workplace climate showed only small correlations with study habits, but showed strong correlations with stress, burnout and satisfaction with medicine. In particular, high stress in the PRHO year showed very significant correlations with measures in 2002 of a perceived high workload, a less supportive-receptive environment, and less choice-independence. In addition, emotional exhaustion both in 2002 and in the PRHO year were related to a high perceived workload in 2002. Personality Table 6 shows the correlations of approaches to work and workplace climate with the 'Big Five' measures of personality, measured both in 2002 and also measured five to six years previously when the doctors were PRHOs. The surface-disordered approach to work is associated with high neuroticism and low conscientiousness, the PRHO correlations also being highly significant in each case. Neuroticism, both in 2002 and as a PRHO, is also associated with a perceived high workload (although in contrast to its prediction of a surface-disordered approach, conscientiousness is not a significant correlate of workload). The deep approach to work and learning is associated with being extravert and with greater openness to experience, and again the measures taken six years earlier are predictive. Finally a supportive-receptive work climate is associated with greater reported agreeableness, both in 2002 and six years earlier as a PRHO. There were no substantial correlations between personality and the surface-rational approach to work or choice-independence in work climate. Multiple regressions Tables 4 to 6 show a large number of correlations, which are not always straightforward to interpret, both because they are numerous and because many variables are themselves inter-correlated. Multiple regression was used to clarify the relationships (for technical details see Supplementary Information see Additional file: 1). Each individual measure of the aAWQ and aWCQ was regressed on the measures of study habits at application (n = 3) and in the final year (n = 3), of stress and burnout during the PRHO year (n = 4) and in 2002 (n = 4), and of personality in the PRHO year (n = 5) and in 2002 (n = 5). Alpha for entry was set at p < 0.0001 in view of the large sample size and the number of independent variables. The variables that were significant are shown in tables 4,5 and 6 and 3 in italics. Of particular interest are variables that show not only show significant contemporaneous correlations but also significant correlations when measured five or more years previously. A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables 4 and 6). The surface-rational approach to work is predicted by strategic learning in medical school, and by less openness to experience and higher conscientiousness. The deep approach to work is predicted by a deep approach to learning at medical school, by greater extraversion, by greater openness to experience, and by lower emotional exhaustion. A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables 5 and 6). A supportive-receptive workplace is predicted by lower stress and depersonalisation, and a higher sense of personal accomplishment when measured previously, and by a more agreeable personality. Choice-independence in the work environment is predicted only by lower previous measures of stress. Stress, burnout and satisfaction with medicine Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table 7 shows the correlations of the five 'stress-related measures' (GHQ, the three burnout measures and satisfaction) with measures of learning style and personality, in each case measured on two separate occasions. Personality correlates with each of the measures, as do study habits. Because of the complex inter-correlations between the dependent variables, multiple regression was used, as before, to find the most important relationships (for technical details see Supplementary Information see Additional file: 1). Doctors who are most stressed showed higher levels of neuroticism, both currently and previously, and those reporting most emotional exhaustion also had higher neuroticism levels, as well as being more introvert. High levels of depersonalisation related to lower levels of agreeableness. A greater sense of personal accomplishment related to previous deep approaches to study and learning, as well as to being more extravert. Overall satisfaction with medicine as a career related to lower levels of neuroticism. Path analysis The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling [20], which analyses the entire set of correlations between variables, using plausible assumptions about causality and removing non-significant paths. The path diagram, which was analysed using LISREL 8.52 [21], is shown in figure 2. Measures to the left can causally influence measures to their right. Based on the time-lagged correlations reported previously, we assumed that stress causes different approaches to work, and we also assumed that approaches to work cause differences in workplace climate rather than vice-versa. (Nevertheless, we acknowledge that the causation may well be reciprocal, as suggested by the originators of the scale [2,19]; further longitudinal data will be required to test that hypothesis). Study habits are temporally and causally prior to stress, approaches to work and workplace climate. Personality, being a trait, was prior to all other measures. For technical details see the Supplementary Information (see Additional file: 1). Although several of our variables are measured at different time points, we have chosen not to present a model in which each variable has been included on each occasion that it is measured, as the resulting diagram becomes unmanageably complex. Although the path diagram in figure 2 is complex at first sight, the paths are readily interpretable. The diagram divides into two broad sections, with the measures of learning style and approach to work at the bottom, and stress at the top. Here we have simplified the model by omitting the closely correlated measures of burnout, and only including paths with t-values greater than 3.6. Estimates of all the paths are available in the Supplementary Information (see Additional file: 1). Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style. Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others [22]. Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience. Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness. Discussion Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric 'caseness'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors "whose stability of character and devotion to duty make one proud of our profession" with those who find it difficult to keep "the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine" [1]. In 2001, Richard Smith asked "Why are doctors so unhappy?" and concluded that "The most obvious cause of doctors' unhappiness is that they feel overworked and undersupported" [23]. Certainly many doctors in our study report a high workload and a work climate that is neither supportive nor receptive, and those doctors also report more stress, burnout and dissatisfaction with medicine as a career. It is tempting therefore to conclude, as did an article in a special edition of BMJ Careers devoted to "Doctors' Wellbeing", that excessive workload and absence of support are directly caused by poor working conditions: "the way in which the NHS is run generates stress for members of the workforce every day" [24]. However, such an interpretation is not straightforward in general [25]. It is particularly difficult for the doctors in our study because the study is longitudinal, and workload and lack of support correlate with stress and burnout reported five or six years earlier, when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of BMJ Careers: "A critical element contributing to the stress that many conscientious doctors experience is internal..." [26]. A similar conclusion was reached in a previous study of ours when these doctors were PRHOs, and multi-level modelling showed that stress is not a characteristic of jobs but of doctors, different doctors working in the same job being no more similar in their stress and burnout than different doctors in different jobs [11]. If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva et al [2] describe as a surface-disorganised approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs. Some doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time – those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30. An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span [27-29] (and across our two measures six years apart), as well as their heritable component [30], means that we have little doubt that personality at selection would also have been predictive, particularly given that a similar pattern of correlations was found in a different cohort of doctors in mid-career [15]. Other studies on very different groups of students have also found, like us, that both strategic and deep learning correlate with conscientiousness, and that deep learning also correlates with extraversion and openness to experience [22,31]. Our study has, for various reasons, not looked at academic performance in relation to study habits, learning styles and personality, although previous work of ours has found clear correlations between learning styles and examination performance [32]. In contrast we have not found any correlation of undergraduate or postgraduate academic achievement with personality [15], and although some studies have found correlations of conscientiousness with academic achievement [33], this does seem to vary according to the learning context [34,35]. Although we will be looking at this question again in more detail in a further analysis, it does seem probable that personality mostly has an indirect effect upon academic achievement via approaches to learning [31,36]. If, as William Wordsworth said, "the child is father to the man", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality [37] and motivation [38], and learning styles with personality [22] . However, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature [39], the environment building upon the genes, and the genes using what is provided by the environment; the poetic complement to William Wordsworth is therefore Alexander Pope, who said, "This education forms the common mind: Just as the twig is bent, the tree's inclined." Extreme introverts can, with sufficient insight, preparation and appropriate training become effective public speakers, less conscientious individuals can learn to be more organised and efficient, and those who are more neurotic can transcend their anxieties (and indeed neuroticism may be beneficial if sublimated into a professional concern for detail in critical situations, rather than merely being undifferentiated personal anxiety). . Formal education, particularly effective formal education [40], can also alter study habits and learning styles, which are less fixed and 'trait-like' than personality measures [17]. Intercalated degrees increase deep and strategic learning and decrease surface learning at medical school [41], making it likely that they also encourage surface-rational and deep approaches to work. Deep and strategic learning also relate to the clinical experience gained by medical students [32], making it possible that greater patient involvement during undergraduate clinical training, rather than mere reliance on textbook learning to pass exams, a characteristic of surface learners, will also reduce surface-disorganised approaches to work. Conclusions Longitudinal data suggest that personality and learning style are not merely correlates of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are causes, events later in time being predicted by events earlier in time [35]. Doctors with greater stress and emotional exhaustion, who were less satisfied with medicine as a career, had higher neuroticism scores and were more likely to be surface-disorganised. Lower conscientiousness on the personality measure also predicted greater stress. Extraverts reported more personal accomplishment and were more satisfied with medicine. The personality measure of agreeableness predicted a more supportive-receptive work environment. These results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions. Competing interests None declared. Authors' contributions The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Click here for file Acknowledgments We thank the many doctors who have participated in this study over the past twelve years. We are also grateful to the three reviewers for their very helpful suggestions. This follow-up of the 1991 cohort was funded by the London Deanery. The survey of applicants was funded by the Leverhulme Trust and the Department of Health, follow-ups during medical school were funded by the Nuffield Foundation and the Department of Health, and the PRHO follow-up was funded by North Thames Postgraduate Medical and Dental Education. Figures and Tables Figure 1 Diagram showing the timing of the various stages of the survey. Note that not all applicants entered in October 1991 (despite in most cases having applied to do so), and subsequent stages of the study therefore took place at different times. Some entrants also took a year longer because of taking an intercalated degree (represented by the diagonal arrow from entrants in Oct 1991 to Final year in 1997), and a few other students delayed for other reasons. All doctors were surveyed at the end of their PRHO year, whenever that had occurred. The cohort was finally brought back into step as a single cohort with the 2002 follow-up when all doctors were studied at the same time, irrespective of the time at which they qualified. Questionnaires were given out at all the boxes shown (with the exception of a questionnaire to Entrants, who are shown merely to make the flow clearer). It should also be noted that there was a follow-up of a subset of the students in their third academic year, which is not shown here because the data are not discussed in this study. Figure 2 Path diagram showing the relationships among the measures of personality, learning style, stress, approaches to work, and workplace climate. The width of arrows is proportional to the strength of an effect, which is shown alongside each line as a path (beta) coefficient. Negative effects are shown as red, dashed lines. For details of the statistical method and a fuller model incorporating all links, see Supplementary Information. Table 1 Dimensions of the AWQ and WCQ [2]. Approaches to work questionnaire Surface disorganised: Feeling overwhelmed by work. For example, being unsure what is needed to complete a task, finding it difficult to organise time effectively, reading things without really understanding them. Surface rational: Preference for order, detail, and routine. For example likes to know precisely what is expected, puts of a lot of effort into memorising important facts when learning something new. Deep approach: Integrative approach that leads to personal understanding. For example, tries to relate new ideas to situations where they might apply. Workplace climate questionnaire Choice-independence: Perception of control over what one does and how one does it. Supportive-receptive: Perception that help is available in the workplace and colleagues are understanding. Workload: Perception of heavy workload and having to cope alone. Table 2 The three separate components of burnout [42]. Note: burnout on the MBI is indicated by higher scores on the emotional exhaustion and depersonalisation scales, and lower scores on the scale of personal accomplishment. Emotional exhaustion: Reduced energy and job enthusiasm; emotional and cognitive distancing from the job. Depersonalisation: Cynicism; lack of engagement and distancing from patients; treatment of patients as inanimate, unfeeling objects. Personal accomplishment: A sense of efficacy and effectiveness; of involvement, commitment and engagement; of capacity to innovate, change and improve. Table 3 Differences in motivation and process of the surface, deep and strategic approaches to learning assessed in the Study Process Questionnaire [41]. Motivation Process Surface Completion of the course Fear of failure Rote learning of facts and ideas Focussing on task components in isolation Little real interest in content Deep Interest in the subject Vocational relevance Personal understanding Relate ideas to evidence Integration of material across courses Identifying general principles Strategic Achieving high grades Competing with others To be successful Use techniques that achieve highest grades Level of understanding Patchy and variable Table 4 Pearson correlation (Significance; N) of background measures with the Approaches to Work Questionnaire. Correlations significant at p < 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are indicated in italics. Time of measurement aAWQ Surface-Disorganised aAWQ Surface-Rational aAWQ Deep aWCQ Workload 2002 0.259 (p < .001; N = 1644) 0.045 (p = .071, N = 1642) -0.068 (p = .006, N = 1638) aWCQ Supportive-Receptive 2002 -0.112 (p < .001; N = 1636) 0.064 (P = .009; N = 1633) 0.102 (P < .001; N = 1630) aWCQ Choice-Independence 2002 -0.125 (P < .001; N = 1647) -0.001 (P = .976; N = 1645) 0.209 (P < .001; N = 1641) SPQ Surface Learning Application 0.119 (P < .001; N = 1591) 0.105 (P < .001; N = 1589) -0.081 (P < .001; N = 1584) Final Year 0.105 (P = .001; N = 999) 0.101 (P = .001; N = 1001) -0.109 (P = .001; N = 996) SPQ Strategic Learning Application -0.101 (P < .001; N = 1591) 0.203 (P < .001; N = 1589) 0.052 (P = .038; N = 1584) Final Year -0.157 (P < .001; N = 999) 0.215 (P < .001; N = 1001) 0.149 (P < .001; N = 996) SPQ Deep Learning Application -0.078 (P = .002; N = 1591) 0.067 (P = .007; N = 1589) 0.192 (P < .001; N = 1584) Final Year -0.131 (P < .001; N = 999) 0.152 (P < .001; N = 1001) 0.240 (P < .001; N = 996) General Health Questionnaire PRHO 0.169 (P < .001; N = 974) 0.010 (P = .761; N = 974) -0.079 (P = .014; N = 972) 2002 0.294 (P < .001; N = 1611) 0.018 (P = .471; N = 1608) -0.119 (P < .001; N = 1604) aMBQ – Emotional exhaustion PRHO 0.062 (P = .103; N = 705) -0.011 (P = .777; N = 704) -0.042 (P = .267; N = 704) 2002 0.223 (P < .001; N = 1609) 0.007 (P = .782; N = 1607) -0.157 (P < .001; N = 1602) aMBQ – Depersonalisation PRHO -0.003 (P = .939; N = 661) -0.033 (P = .400; N = 658) -0.017 (P = .672; N = 659) 2002 0.224 (P < .001; N = 1598) -0.071 (P = .005; N = 1596) -0.151 (P < .001; N = 1590) aMBQ – Personal accomplishment PRHO -0.137 (P < .001; N = 938) -0.032 (P = .335; N = 937) 0.156 (P < .001; N = 936) 2002 0.024 (P = .335; N = 1604) -0.013 (P = .610; N = 1602) 0.095 (P < .001; N = 1596) Satisfaction with medicine as a career 2002 -0.162 (P < .001; N = 1620) 0.037 (P = .135; N = 1617) 0.211 (P < .001; N = 1612) Table 5 Pearson correlation (Significance; N) of background measures with the Workplace Climate Questionnaire. Correlations significant at p < 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in italics. Time of measurement aWCQ Workload aWCQ Supportive-Receptive aWCQ Choice-Independence SPQ Surface Learning Application 0.022 (P = .389; N = 1587) 0.042 (P = .093; N = 1578) 0.004 (P = .874; N = 1591 Final Year 0.037 (P = .240; N = 999) -0.034 (P = .287; N = 998) 0.054 (P = .090; N = 1000) SPQ Strategic Learning Application 0.032 (P = .196; N = 1591) 0.043 (P = .090; N = 1578) 0.082 (P = .001; N = 1591) Final Year 0.063 (P = .048; N = 999) 0.020 (P = .520; N = 998) 0.036 (P = .255; N = 1000) SPQ Deep Learning Application 0.008 (P = .738; N = 1587) 0.029 (P = .251; N = 1578) 0.049 (P = .051; N = 1591) Final Year -0.032 (P = .306; N = 999) 0.071 (P = .026; N = 998) 0.065 (P = .041; N = 1000) General Health Questionnaire PRHO 0.183 (P < .001; N = 973) -0.134 (P < .001; N = 970) -0.110 (P = .001; N = 972) 2002 0.354 (P < .001; N = 1606) -0.239 (P < .001; N = 1597) -0.259 (P < .001; N = 1609) aMBQ – Emotional exhaustion PRHO 0.211 (P < .001; N = 703) -0.056 (P = .136; N = 701) -0.035 (P = .348; N = 703) 2002 0.400 (P < .001; N = 1607) -0.164 (P < .001; N = 1607) -0.220 (P < .001; N = 1609) aMBQ – Depersonalisation PRHO 0.057 (P = .145; N = 658) -0.109 (P = .005; N = 656) -0.020 (P = .609; N = 658) 2002 0.216 (P < .001; N = 1595) -0.222 (P < .001; N = 1594) -0.165 (P < .001; N = 1597) aMBQ – Personal accomplishment PRHO -0.028 (P = .393; N = 937) 0.110 (P < .001; N = 933) 0.063 (P = .055; N = 936) 2002 0.163 (P < .001; N = 1601) 0.143 (P < .001; N = 1600) 0.044 (P = .079; N = 1603) Satisfaction with medicine as a career 2002 -0.256 (P < .001; N = 1617) 0.317 (P < .001; N = 1616) 0.294 (P < .001; N = 1620) Table 6 Pearson correlation (Significance; N) of Approach to Work Questionnaire and the Workplace Climate Questionnaire with personality. Correlations significant at p < 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in shaded boxes. Time of measurement aAWQ Surface-Disorganised aAWQ Surface-Rational aAWQ Deep aWCQ Workload aWCQ Supportive-Receptive aWCQ Choice-Independence Neuroticism PRHO 0.172 (P < .001; N = 993) 0.021 (P = .510; N = 993) -0.050 (P = .113; N = 992) 0.111 (P < .001; N = 992) -0.055 (P = .086; N = 989) -0.017 (P = .592; N = 991) 2002 0.335 (P < .001; N = 1645) 0.021 (P = .405; N = 1643) -0.111 (P < .001; N = 1638) 0.307 (P < .001; N = 1642) -0.180 (P < .001; N = 1632) -0.168 (P < .001; N = 1644) Extraversion PRHO -0.113 (P < .001; N = 991) 0.056 (P = .079; N = 991) 0.174 (P < .001; N = 989) -0.090 (P = .004; N = 990) -0.087 (P = .006; N = 987) 0.049 (P = .124; N = 989) 2002 0.173 (P < .001; N = 1649) 0.037 (P = .133; N = 1647) 0.288 (P < .001; N = 1643) -0.164 (P < .001; N = 1647) 0.147 (P < .001; N = 1637) 0.119 (P < .001; N = 1649) Openness to experience PRHO -0.011 (P = .729; N = 975) -0.077 (P = .016; N = 975) 0.263 (P < .001; N = 973) -0.037 (P = .250; N = 974) 0.036 (P = .259; N = 971) 0.033 (P = .297; N = 973) 2002 -0.063 (P = .010; N = 1646) -0.108 (P < .001; N = 1644) 0.346 (P < .001; N = 1639) -0.096 (P < .001; N = 1643) 0.038 (P = .129; N = 1633) 0.080 (P < .001; N = 1645) Agreeableness PRHO -0.034 (P = .285; N = 991) 0.048 (P = .130; N = 991) 0.038 (P = .235; N = 989) -0.024 (P = .455; N = 990) 0.182 (P < .001; N = 987) 0.026 (P = .421; N = 989) 2002 -0.108 (P < .001; N = 1650) 0.096 (P < .001; N = 1648) 0.038 (P = .121; N = 1643) -0.057 (P = .021; N = 1647) 0.153 (P < .001; N = 1638) 0.017 (P = .498; N = 1650) Conscientiousness PRHO -0.353 (P < .001; N = 991) 0.124 (P < .001; N = 991) 0.084 (P = .008; N = 989) 0.023 (P = .467; N = 991) 0.057 (P = .074; N = 987) 0.056 (P = 077; N = 990) 2002 -0.477 (P < .001; N = 1645) 0.126 (P < .001; N = 1643) 0.136 (P < .001; N = 1638) -0.088 (P < .001; N = 1641) 0.093 (P < .001; N = 1632) 0.092 (P < .001; N = 1644) Table 7 Pearson correlation (Significance; N) of stress, burnout and satisfaction with medicine as a career in 2002 with study habits at application to medical school and in th final year of medical school, and with personality in the PRHO year and in 2002. Correlations significant at p < 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in shown in italics. Time of measurement Stress (GHQ) (2002) Emotional exhaustion (aMBI) (2002) Depersonalisation (aMBI) Personal accomplishment (aMBI) Satisfaction with medicine as a career SPQ Surface Learning Application 0.036 (P = .150; N = 1555) 0.105 (P < .001;N = 1552) 0.068 (P = .008;N = 1542) -0.003 (P = .895;N = 1546) -0.033 (P = .186;N = 1563) Final Year 0.008 (P = .792;N = 984) 0.090 (P = .005;N = 983) 0.094 (P = .003;N = 976) -0.036 (P = .266;N = 980) -0.023 (P = .471;N = 989) SPQ Strategic Learning Application -0.011 (P = .671; N = 1555) 0.017 (P = .513;N = 1552) -0.073 (P = .004;N = 1542) 0.021 (P = .416;N = 1546) 0.046 (P = .068;N = 1563) Final Year -0.014 (P = .655;N = 984) -0.052 (P = .102;N = 983) -0.108 (P = .001;N = 976) 0.007 (P = .819;N = 980) 0.104 (P = .001;N = 989) SPQ Deep Learning Application 0.041 (P = .102; N = 1555) -0.001 (P = .974;N = 1552) -0.055 (P = .031;N = 1542) 0.123 (P < .001;N = 1546) 0.042 (P = .101;N = 1563) Final Year -0.022 (P = .498;N = 984) -0.105 (P = .001;N = 983) -0.071 (P = .027;N = 976) 0.120 (P < .001;N = 980) 0.134 (P < .001;N = 989) Neuroticism PRHO 0.192 (P < .001;N = 972) 0.233 (P < .001;N = 972) 0.103 (P = .001;N = 972) 0.036 (P = .255;N = 975) -0.188 (P < .001;N = 981) 2002 0.461 (P < .001; N = 1610) 0.378 (P < .001;N = 1607) 0.235 (P < .001;N = 1596) 0.090 (P < .001;N = 1603) -0.314 (P < .001;N = 1618) Extraversion PRHO -0.111 (P = .001;N = 970) -0.190 (P < .001;N = 970) -0.137 (P < .001;N = 969) 0.077 (P = .016;N = 972) 0.228 (P < .001;N = 979) 2002 -0.243 (P < .001; N = 1614) -0.262 (P < .001;N = 1611) -0.171 (P < .001;N = 1599) 0.152 (P < .001;N = 1606) 0.307 (P < .001;N = 1621) Openness to experience PRHO 0.012 (P = .721;N = 956) 0.017 (P = .604;N = 955) 0.009 (P = .777;N = 954) 0.096 (P = .003;N = 957) 0.042 (P = .194;N = 964) 2002 -0.046 (P = .066; N = 1611) 0.010 (P = .677;N = 1607) -0.028 (P = .262;N = 1596) 0.127 (P < .001;N = 1603) 0.066 (P = .008;N = 1618) Agreeableness PRHO -0.028 (P = .376;N = 970) -0.062 (P = .055;N = 970) -0.240 (P < .001;N = 969) 0.091 (P = .005;N = 972) 0.101 (P = .002;N = 979) 2002 -0.080 (P = .001; N = 1615) -0.094 (P < .001;N = 1611) -0.322 (P < .001;N = 1600) 0.082 (P = .001;N = 1607) 0.135 (P < .001;N = 1622) Conscientiousness PRHO -0.045 (P = .165;N = 971) -0.088 (P = .006;N = 970) -0.121 (P < .001;N = 969) 0.010 (P = .767;N = 972) 0.045 (P = .160;N = 979) 2002 -0.196 (P < .001; N = 1610) -0.129 (P < .001;N = 1606) -0.165 (P < .001;N = 1595) 0.031 (P = .209;N = 1602) 0.162 (P < .001;N = 1617) ==== Refs Osler W Aequanimitas 1932 3 London: H K Lewis Delva MD Kirby JR Knapper CK Birtwhistle RV Postal survey of approaches to learning among Ontario physicians: implications for continuing medical education Brit Med J 2002 325 1218 12446540 10.1136/bmj.325.7374.1218 Knapper CK Gibbs C Approaches to study and lifelong learning: Some Canadian initiatives In Improving student learning through assessment and evaluation 1995 Oxford: Oxford Centre for Staff Development Kirby JR Knapper CK Evans CJ Carty AE Gadula C Approaches to learning at work and workplace climate International Journal of Training and Development 2003 7 31 52 10.1111/1468-2419.00169 Delva MD Schultz K Kirby J Godwin M Learning approaches in clerkship and residency: The effect of workplace climate Submitted 2003 Deary IJ Blenkin H Agius RM Endler NS Zealley H Wood R Models of job-related stress and personal achievement among consultant doctors British Journal of Psychology 1996 87 3 29 8852018 Entwistle N Styles of learning and teaching 1981 Chichester: John Wiley Newble DI Entwistle NJ Learning styles and approaches: implications for medical education Med Educ 1986 20 162 175 3724571 McManus IC Richards P Winder BC Sproston KA Styles V Medical school applicants from ethnic minorities: identifying if and when they are disadvantaged Brit Med J 1995 310 496 500 7888888 McManus IC Factors affecting likelihood of applicants being offered a place in medical schools in the United Kingdom in 1996 and 1997: retrospective study Brit Med J 1998 317 1111 1116 9784446 McManus IC Winder BC Paice E How consultants, hospitals, trusts and deaneries affect pre-registration house officer posts: a multilevel model Med Educ 2002 36 35 44 11849522 10.1046/j.1365-2923.2002.01123.x Goldberg DP The detection of psychiatric illness by questionnaire 1972 London: Oxford University Press Maslach C Jackson SE Maslach Burnout Inventory 1986 Palo Alto, CA: Consulting Psychologists Press McManus IC Winder BC Gordon D The causal links between stress and burnout in a longitudinal study of UK doctors Lancet 2002 359 2089 2090 12086767 10.1016/S0140-6736(02)08915-8 McManus IC Smithers E Partridge P Keeling A Fleming PR A levels and intelligence as predictors of medical careers in UK doctors: 20 year prospective study Brit Med J 2003 327 139 142 12869457 10.1136/bmj.327.7407.139 Biggs JB Study process questionnaire: Manual 1987 Melbourne: Australian Council for Educational Research Fox RA McManus IC Winder BC The shortened Study Process Questionnaire: an investigation of its structure and longitudinal stability using confirmatory factor analysis British Journal of Educational Psychology 2001 71 511 530 11802815 10.1348/000709901158659 Matthews G Deary IJ Personality traits 1998 Cambridge: Cambridge University Press Kirby JR Delva MD Knapper CK Birtwhistle RV Development of the Approaches to Work and Workplace Climate Questionnaires for Physicians Evaluation and the Health Professions 2003 26 104 121 10.1177/0163278702250097 Maruyama GM Basics of structural equation modelling 1998 Thousand Oaks, California: Sage Jöreskog KG Sörbom D LISREL 8 User's Reference Guide 1993 Chicago, IL: Scientific Software International Zhang L-F Does the big five predict learning approaches? Personality and Individual Differences 2003 34 1431 1446 10.1016/S0191-8869(02)00125-3 Smith R Why are doctors so unhappy? Brit Med J 2001 322 1073 1074 11337419 10.1136/bmj.322.7294.1073 Chambers R Supporting GPs Brit Med J 2003 326 s100 12663426 10.1136/bmj.326.7391.S100 Dollard MF Dollard MF, Winefield AH, Winefield HR Introduction: Context, theories and intervention In Occupational stress in the service professions 2003 London: Taylor and Francis 1 42 Menninger R Stress: defining the personal equation Brit Med J 2003 326 s107 s108 12663432 10.1136/bmj.326.7391.S107 Matthews G Deary IJ Whiteman MC Personality traits 2003 second Cambridge: Cambridge University Press Roberts BW DelVecchio WF The rank-order consistency of personality traits from childhood to old age: A quantitative review of longitudinal studies Psychol Bull 2000 126 3 25 10668348 10.1037//0033-2909.126.1.3 McCrae RR Costa PT Jr Personality in adulthood: A five-factor theory perspective 2003 2 New York: Guilford Press Loehlin JC Genes and environment in personality development 1992 Newbury Park, CA: Sage Duff A Boyle E Dunleavy K Ferguson J The relationship between personality, approach to learning and academic performance Personality and Individual Differences 2004 36 1907 1920 10.1016/j.paid.2003.08.020 McManus IC Richards P Winder BC Sproston KA Clinical experience, performance in final examinations, and learning style in medical students: prospective study Brit Med J 1998 316 345 350 9487168 Ferguson E James D Madeley L Factors associated with success in medical school and in a medical career: systematic review of the literature Brit Med J 2002 324 952 957 11964342 10.1136/bmj.324.7343.952 Ferguson E James D O'Hehir F Saunders A A pilot study of the roles of personality, references and personal statements in relation to performance over the five years of a medical degree Brit Med J 2003 McManus IC Commentary: How to derive causes from correlations in educational studies Brit Med J 2003 326 432 Ramsden P Learning to teach in higher education 1992 London: Routledge Hurtz GM Donovan JJ Personality and job performance: The Big Five revisited J App Psych 2000 85 869 879 10.1037//0021-9010.85.6.869 Barrick MR Stewart GL Piotrowski M Personality and job performance: test of the mediating effects of motivation among sales representatives J App Psych 2002 87 43 51 10.1037//0021-9010.87.1.43 Ridley M Nature via Nurture: Genes, experience and what makes us human 2003 London: Fourth Estate Biggs JB Teaching for quality learning at university 2003 Milton Keynes: SRHE Open University Press McManus IC Richards P Winder BC Intercalated degrees, learning styles, and career preferences: prospective longitudinal study of UK medical students Brit Med J 1999 319 542 546 10463892 Maslach C Schaufeli WB Leiter MP Job burnout Annual Review of Psychology 2001 52 397 422 11148311 10.1146/annurev.psych.52.1.397

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==== Front Theor Biol Med ModelTheoretical Biology & Medical Modelling1742-4682BioMed Central London 1742-4682-1-71533934710.1186/1742-4682-1-7ResearchCould a simple surgical intervention eliminate HIV infection? Tepic Slobodan [email protected] School of Veterinary Medicine, University of Zurich, Zurich, Switzerland2004 31 8 2004 1 7 7 3 8 2004 31 8 2004 Copyright © 2004 Tepic; licensee BioMed Central Ltd.2004Tepic; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Human Immunodeficiency Virus (HIV) infection is a dynamic interaction of the pathogen and the host uniquely defined by the preference of the pathogen for a major component of the immune defense of the host. Simple mathematical models of these interactions show that one of the possible outcomes is a chronic infection and much of the modelling work has focused on this state. Bifurcation However, the models also predict the existence of a virus-free equilibrium. Which one of the equilibrium states the system selects depends on its parameters. One of these is the net extinction rate of the preferred HIV target, the CD4+ lymphocyte. The theory predicts, somewhat counterintuitively, that above a critical extinction rate, the host could eliminate the virus. The question then is how to increase the extinction rate of lymphocytes over a period of several weeks to several months without affecting other parameters of the system. Testing the hypothesis Proposed here is the use of drainage, or filtration, of the thoracic duct lymph, a well-established surgical technique developed as an alternative for drug immunosuppression for organ transplantation. The performance of clinically tested thoracic duct lymphocyte depletion schemes matches theoretically predicted requirements for HIV elimination. ==== Body Dynamics of HIV infection and selection of equilibrium states Reports on the high turnover rates of HIV and its preferred target, CD4+ lymphocytes, during the latent phase of HIV infection [1-3] have established the virus as a prime suspect for direct demolition of the immune system. These clinical findings have also stimulated further efforts at modeling [4,5], and quantitative experimental observation [6]. Major journals have a preference for experimental or clinical data, and the results of mathematical modelling have not reached the broader AIDS research community. For example, the most interesting result of a simple dynamic model published several years ago [7], namely the existence of multiple equilibrium states, one of which is virus-free, has not been discussed in any of the recent publications on HIV response to anti-viral drugs. For a general medical audience it would be desirable to describe the basic features of the dynamics of HIV infection without recourse to any mathematical formulations. Dynamics implies change over time and the behavior of a dynamic system is defined by stating how the system variables affect each other during a unit of time. In the simplest model there are three system variables: (i) the number of uninfected lymphocytes, (ii) the number of infected lymphocytes and (iii) the number of free virions. The system equations describe how these populations interact. For HIV/CD4+, some of these interactions are understood and generally accepted; others are more speculative, and are subject to further study. However, even with different assumptions about these lesser known aspects, the most interesting result is little affected because it derives from the fact that the rate of infection, i.e. the number of newly infected cells in a unit of time, is proportional to the product of the number of uninfected cells and the number of free virions. This makes the resulting equations non-linear, and when the question of equilibrium is addressed, which is done by setting all rates (changes with time) equal to zero, there are two distinct solutions. One of these is free of virus, i.e. the number of virions (and infected cells) is equal to zero, whilst the other equilibrium state has non-zero values for all three populations and thus corresponds to a chronic infection. Which one of the two equilibrium states the system attains depends on the values of the system parameters. The most natural parameter to consider for switching the states is the difference between the rates at which uninfected cells are dying and proliferating. If this parameter, the net extinction rate of healthy lymphocytes, is increased above a critical value, the virus-free equilibrium is selected. This selection (bifurcation) is driven by the conditions of stability; the chronic infection state becomes unstable, i.e. any disturbance takes the system out of it, whilst the virus-free state becomes stable. Once the net extinction rate exceeds the critical value, the system finds its way out of infection. It just so happens that the amount by which the extinction rate needs to be changed, and this based on our current best estimates of other values, is quite modest – several percent of the total lymphocyte population needs to be removed daily. Depletion of lymphocytes as a therapy for AIDS, based on a population dynamic model, has been advocated by de Boer and Boucher [8]. They proposed that using a suitable immunosuppressant or CD4-killing drug in combination with an anti-viral therapy may eliminate the infection. This author has arrived at the same result independently using a population dynamics model (three populations, as described above), but also using an expanded model that includes the immune response and, in particular, Tat protein-induced apoptosis [9]. The intervention by lymphocyte depletion will work as predicted by modelling only if other parameters of the system remain substantially unaffected. This is an unlikely outcome with immunosuppressive drugs. Results from limited attempts to use them in HIV-positive patients [10-12] are interesting, but not very encouraging. In fact, the observed rise in CD4+ counts runs contrary to the expected effect of depletion. Activation of latent CD4+ by OKT3 and IL-2 with intention to purge the virus has also been attempted [13], but the outcome was a surprisingly prolonged depletion of CD4+ with little effect on the virus. Our knowledge of the immune system interactions seems inadequate to provide satisfactory explanations for such a response. As a further illustration of how complex different interventions with biological modifiers can be, treatments with depleting CD4 monoclonal antibody showed a preferential loss of naive T cells, but did not affect IFN-gamma secreting cells [14], providing a clue as to why such depletions did not meet expectations in treating autoimmune diseases. Depletion of lymphocytes from the lymphatic circulation The prediction of the theoretical model calls for the removal of 5 to 10 percent of the total lymphocyte pool per day. The critical value is subject to uncertain estimates of some parameters, and it does differ between the simple, three-parameter and the five-parameter, expanded model. Perhaps the best approach would be to begin depletion, monitor the response by the viral RNA, and then adjust the depletion rate. All of this suggests that some means of physical removal would be best suited. Extracorporeal blood cell separation is a possibility, but the estimate of several hours that the patient would have to spend on the machine daily for several weeks to months, is very discouraging. However, filtration of the thoracic duct lymph, where lymphocytes are present in high concentration, seems almost ideal. The technique of duct drainage for lymphocyte depletion was developed in the sixties and the seventies in order to reduce the risk of organ rejection [15-21]. It has found fairly broad acceptance in renal transplantation [22-25] where the patients would typically be treated for four weeks prior to receiving a transplant. With improved techniques of tissue matching and better immunosuppressive drugs, the thoracic duct drainage lost its appeal in transplant surgery, but it remains an interesting approach to treatments of autoimmune conditions such as rheumatoid arthritis (RA) [26-28]. Improvements in the biocompatibilty of implants could ostensibly even extend the impressive performance of access devices that have remained potent for hundreds of days [29]. The number of lymphocytes removed from RA patients by thoracic duct filtration [29] is in the range of modelling predictions for elimination of the virus (on the order of ten billion per day at the start of the treatment). An alternative to removal of lymphocytes by duct drainage or filtration is their diversion from the lymphatic system into the gastro-intestinal tract, which has been demonstrated in experimental animals [30-32]. Cells are killed while the precious protein is recycled, avoiding the problem of protein loss by drainage. There is no evidence that HIV could survive gastric passage. The drawback of such a procedure would be in the difficulty of controlling the number of lymphocytes removed. This may not be such a serious limitation, provided the critical value is exceeded. The rate of lymphocyte removal then simply determines the duration of the treatment and the reduction in the number of lymphocytes the patient will experience. This, of course, is an issue that needs careful consideration. Depletion of lymphocytes will cause a transient reduction of their pool (with counts predicted to drop to a few hundred CD4 lymphocytes/microliter), and thus affect the general immune competence. The intervention by depletion should be done as early as possible when the rates of removal necessary are lower and the total pool is less reduced. Concluding remarks Unfortunately, theoretical predictions of system dynamics are not very encouraging for the prospects of HIV vaccines. In principle, the vaccination primes the system for a faster, stronger response, including proliferation of the responding lymphocytes. As the same cells are targets for the virus, the system moves away from the stability condition for the virus-free equilibrium. Apoptosis of uninfected CD4+ lymphocytes in HIV infection is an appropriate response (for the host), albeit insufficient, since the cause of apoptosis is Tat protein produced by only the infected cells themselves. This prevents the system from eliminating the virus because the apoptotic signal weakens along with the infection. This suggests a possibility for a pharmacological intervention based on Tat protein that could sustain the apoptotic signal without introducing molecular modifiers with potentially broader effects. If vaccinations were to work, upon entry of the pathogen, they should provoke apoptosis of lymphocytes, not their proliferation. Until such discoveries are made, and to test perhaps their ultimate potential, a simple surgical intervention to allow for removal of lymphocytes merits further investigation. Dangers posed to the patient would be significant, both due to morbidity of the procedure itself, and the expected, but difficult to precisely predict cumulative effects on immunocompetence. An attractive aspect of using physical means of depletion is the possibility to terminate the treatment instantly and completely, as soon as any major deviations from the expected response would arise, indicating a failure of the model and alarming for unexpected risks. Competing interests None declared. ==== Refs Wei X Ghosh SK Taylor ME Johnson VA Emini EA Deutsch P Lifson JD Bonhoeffer S Nowak MA Hahn BH Saag MS Shaw GM Viral dynamics in human immunodeficiency virus type 1 infection Nature 1995 373 117 122 7529365 10.1038/373117a0 Ho DD Neumann AU Perelson AS Chen W Leonard JM Markowitz M Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection Nature 1995 373 123 126 7816094 10.1038/373123a0 Nowak MA Bonhoeffer S Loveday C Balfe P Semple M Kaye S Tenant-Flowers M Tedder R HIV results in the frame. Results confirmed Nature 1995 375 193 193 7746311 10.1038/375193a0 Coffin JM HIV population dynamics in vivo; implications for genetic variation, pathogenesis, and therapy Science 1995 267 483 489 7824947 Phillips AN Reduction of the HIV concentration during acute infection: independence from a specific immune response Science 1996 271 497 499 8560262 Perelson AS Neumann AU Markowitz M Leonard JM Ho DD HIV-1 dynamics in vivo; virion clearance rate, infected cell life-span, and viral generation time Science 1996 271 1582 1586 8599114 Perelson AS Kirschner D de Boer R Dynamics of HIV infection of CD4+ T cells Math Biosci 1993 114 81 125 8096155 10.1016/0025-5564(93)90043-A de Boer RJ Boucher CAB Anti-CD4 therapy for AIDS suggested by mathematical models Proc R Soc Lond B 1996 263 889 905 Li CJ Friedman DJ Wang C Metelev V Pardee AB Induction of apoptosis in uninfected lymphocytes by HIV-l Tat protein Science 1995 268 429 431 7716549 Andrieu JM Even P Venet A Effects of cyclosporin on T-cell subsets in human immunodeficiency virus disease Clin Immunol Immunopathol 1988 46 181 198 3258211 10.1016/0090-1229(88)90071-2 Andrieu JM Lu W Levy R Sustained increases in CD4 cells counts in asymptomatic human immunodeficiency virus type 1-seropositive patients treated with predinisolone for 1 year J Infect Dis 1995 171 523 530 7876597 Corey L Editorial: reducing T cell activation as a therapy for human immunodeficiency virus infection J Infect Dis 1995 171 521 522 7876596 van Praag RM Prins JM Roos MT Schellekens PT Ten Berge IJ Yong SL Schuitemaker H Eerenberg AJ Jurriaans S de Wolf F Fox CH Goudsmit J Miedema F Lange JM OKT3 and IL-2 Treatment for Purging of the Latent HIV-1 Reservoir in Vivo Results in Selective Long-Lasting CD4+ T Cell Depletion J Clin Immunol 2001 21 218 226 11403229 10.1023/A:1011091300321 Rep MH van Oosten BW Roos MT Ader HJ Polman CH van Lier RA Treatment with Depleting CD4 Monoclonal Antibody Results in a Preferential Loss of Circulating Naive T Cells but does not Affect IFN-Gamma Secreting TH1 Cells in Humans J Clin Invest 1997 99 2225 31 9151795 Joel DD Sautter JH Preparation of a chronic thoracic duct venous shunt in calves Proc Soc Exp 1963 112 856 859 Biol Med Woodruff MFA Anderson NF The effect of lymphocyte depletion by thoracic duct fistula and administration of antilympocytic serum on the survival of skin homografts in rats Ann NY Acad Sci 1964 120 119 14233856 Tilney NL Murray LE Chronic thoracic duct fistula: Operative technic and physiologic effects in man Ann Surg 1968 167 1 4865020 Murray JE Wilson RE Tilney NL Merrill JP Cooper WC Birtch AG Carpenter CB Hager EB Dammin GJ Harrison JH Five years' experience in renal transplantation with immunosuppresive drugs: survival, function, complications, and the role of lymphocyte depletion by thoracic duct fistula Ann Surg 1968 168 416 435 4175449 Fish JC Mattingly AT Ritzmann SE Sarles HE Remmers AR Jr Circulating lymphocyte depletion in the calf. Effect on blood and lymph lymphocytes Arch Surg 1969 99 664 668 5350817 Sharbaugh RJ Fitts CT Majeski JA Wright FA Hargest TS The efficacy of closed-circuit extracorporeal filtration of thoracic duct lymph as a means of lymphocyte depletion Clin exp Immunol 1972 12 255 262 4648823 Majeski JA Fitts CT Sharbaugh RJ Architectural alteration of lymphatic tissue produced by extracorporeal thoracic duct filtration J Surg Oncol 1977 9 155 162 301218 Starzl TE Weil R 3rdKoep LJ McCalmon RT JrTerasaki PI Iwaki Y Schroter GP Franks JJ Subryan V Halgrimson CG Thoracic duct fistula and renal transplantation Ann Surg 1979 190 474 486 384943 Fish JC Sarles HE Remmers A JrTownsend CM JrBell JD Flye MW Renal transplantation after thoracic duct drainage Ann Surg 1981 193 752 756 7018426 Starzl TE Klintmalm GB Iwatsuki S Weil R 3d Late follow-up after thoracic duct drainage in cadaveric renal transplantation Surg Gynecol Obstet 1981 153 377 382 7022708 Takeuchi N Ohshima S Ono Y Sahashi M Matsuura O Yamada S Tanaka K Kuriki O Kamihira O Five-year results of thoracic duct drainage in living related donor kidney transplantation Transplant Proc 1992 24 1421 1423 1496604 Paulus HE Machleder HI Levine S Yu DT MacDonald NS Lymphocyte involvement in rheumatoid arthritis. Studies during thoracic duct drainage Arthritis Rheum 1977 20 1249 1262 302707 Vaughan JH Fox RI Abresch RJ Tsoukas CD Curd JG Carson DA Thoracic duct drainage in rheumatoid arthritis Clin Exp Immunol 1984 58 645 653 6334581 Sany J Immunological treatment of rheumatoid arthritis Clin Exp Rheumatol 1990 8 81 88 2245532 Sato T Koga N Nagano T Ohteki H Masuda T Agishi T Improved on-line thoracic duct drainage for lymphocytapheresis Int J Artif Organs 1991 14 800 804 1783455 Flintoff WM JrTucker HM Increased homograft survival. Internal thoracic duct-esophageal shunt Arch Otolaryngol 1973 97 251 252 4572183 Kawai T Stoitchcov E Lorenzini C Merle M Benichoux R Long-term follow-up of dogs, with a patent anastomosis of the thoracic duct to the esophagus Eur Surg Res 1974 6 46 55 4853034 Williamson EP Sells RA The chyloesophageal fistula. A new approach to thoracic duct drainage Transplantation 1986 42 136 140 3526653

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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-221531765910.1186/1742-4690-1-22ReviewThe retroviral RNA dimer linkage: different structures may reflect different roles Greatorex Jane [email protected] Division of Infectious Diseases, Dept. of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK2004 18 8 2004 1 22 22 10 8 2004 18 8 2004 Copyright © 2004 Greatorex; licensee BioMed Central Ltd.2004Greatorex; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Retroviruses are unique among virus families in having dimeric genomes. The RNA sequences and structures that link the two RNA molecules vary, and these differences provide clues as to the role of this feature in the viral lifecycles. This review draws upon examples from different retroviral families. Differences and similarities in both secondary and tertiary structure are discussed. The implication of varying roles for the dimer linkage in related viruses is considered. ==== Body Introduction With relatively few genes compared to many other virus families, the retroviridae have evolved over the millenia to maximise the functions of their RNA genome. The genome serves as a versatile template from which various proteins can be translated by the use of splicing and by translational flexibility using scanning, IRES and frameshifting. It is also an RNA molecule capable of interacting with itself, and cellular and viral proteins. By these means, from an RNA around 7 – 12 kilobases long, the retroviridae have evolved to infect a wide range of species and cell types. A unique characteristic of retroviral genomes is the fact that they are dimeric. The reasons for this are as yet unclear, and are discussed below. In brief, it is thought that the diploid genome allows template switching during reverse transcription and may be linked to recombination in some viruses. It may also play a role in translation of proteins and packaging of the RNA. Much of the work on the nature, structure(s), and role of the dimer linkage has been based on Human Immunodeficiency Virus Type 1, and this has been recently reviewed ([1] and Russell et al this issue [2]). Whether or not HIV-1 is a representative model for other retroviruses is open to debate. However, there have been important contributions from investigators studying other retroviruses. They have shown both similarities with the HIV-1 motifs, and also, importantly, differences. The fact that distinct RNA structures are used by different retroviruses to perform the same purpose, namely to link their two RNA molecules, tells us something very important. For these viruses, whatever organism or cell they are infecting it has been advantageous to evolve to do so with a double complement of genome in their virion particles. However, diploidy may be used to benefit the virus in a number of ways and for different viruses the priorities may vary. This review will attempt to draw on several examples from viruses other than HIV-1, whilst of necessity drawing comparisons with the latter. The dimeric genome Retroviruses were discovered at the beginning of the 20th century [3,4]. The unique nature of their genome was first discovered in the 1960s [5,6] but the actual dimeric genomes were elucidated, and visualised by electron microscopy, a decade later [7,8]. Bender and colleagues extracted the RNA from several different retroviruses and examined it by electron microscopy under denaturing conditions. The RNA appeared to be joined at a discrete point, termed the dimer linkage site (DLS). Using bromodU to label the RNA at one end, they were able to show that the molecules were joined at their 5' ends [9,10]. Under less stringent conditions the genomes can be demonstrated to interact along their lengths [11] and it is this that probably contributes to confusing reports on the exact location of the primary DLS in different viruses. RNA dimerisation in the primate lentiviruses, predominantly HIV-1, has subsequently been extensively studied [1], yet little has been published on this process in the non-primate lentiviruses. Early studies of rapid harvest virions of the prototype lentivirus, Maedi Visna virus (MVV), identified viral RNA with a Svedberg coefficient of 35S immediately post-budding, which increased with time to 70S. It is possible that weakly interacting dimers formed during RNA encapsidation may have been denatured during purification, however these observations are supportive of a progression from monomeric to dimeric RNA associated with viral maturation [12]. Since 1990 [13] it has been possible to study in vitro the RNA elements involved in the dimer linkage first observed by EM. It was shown that RNA transcripts comprising sequences from the 5' end of the viral genome would migrate as two species of RNA when subjected to electrophoresis. By this means many subsequent studies were able to focus on isolating the elements and structures involved in dimerisation, and to investigate the role of the viral structural proteins in this process. Multiple functions for the dimeric genome? As yet investigators have not been able to agree on a distinct role for the dimer linkage. The fact that it is conserved amongst the retroviridae does not guarantee that its role will be the same in all retroviral families. The following section of the review will endeavour to explore some of the proposed roles, and examine the evidence from different retroviruses. The dimeric linkage and recombination Several studies have demonstrated that, in HIV-1 and MLV, the dimer linkage serves as a "hotspot" for recombination [14,15]. It is an obvious hypothesis, that in viruses which are known for their hypervariability, there exists the capacity to jump from one RNA molecule to another. Researchers have compared dimerising to non-dimerising controls, and the frequency and distribution of template switching. Templates containing the dimerisation site had a 4-fold higher transfer efficiency than the non-dimerising control [14]. This result implies that recombination would occur preferentially at the site where the RNA molecules were in close proximity. In the case of HIV-1, whilst it has been shown that template switching is facilitated by template homology [16], it has also been demonstrated that recombination can occur between viruses of different subtypes which might have different dimer initiation sequences (DIS) [17]. Bearing in mind the fact that the genome is linked at other sites besides the DIS [11], it seems probable that other hot spots for recombination exist. Interestingly, it has been suggested that the nucleocapsid protein (NC) promotes or stimulates the strand transfer reaction. As will be discussed below, NC and the precursor Gag protein both bind the RNA close to the DIS in HIV-1. In addition, there is evidence that the presence of a dimer in the virus particle facilitates the first strand-transfer reaction of reverse transcription [18]. Work in our laboratory has shown that the Maedi Visna Virus DIS is centred on a helix terminating in a GACG tetraloop between positions 281 and 300 in the viral genome; a region which is highly conserved between the ovine and caprine lentiviruses (Monie, personal communication, see Figure 3d). Intriguingly, this structure shows homology with structural motifs in the Alpha- and Gammaretroviruses, but not with DIS regions identified in the primate lentiviruses. Within the Alpha- and Gammaretroviruses GACG tetraloops are involved in the packaging of viral RNA [19,20] and whilst not a component of the core M-MLV DIS motif [21], they may contribute to the process of dimerisation and the stability of the resultant dimer [22]. Importantly, it is possible to form heterodimers between transcripts from these viruses containing the GACG tetraloops and between MVV and M-MLV (personal observations). This raises parallels with recent studies of the dimerisation of murine leukaemia viruses and Harvey Sarcoma virus in which GACG tetraloops were found to regulate inter-species RNA heterodimerisation [23], whilst other linkage elements were postulated to mediate homodimerisation. Recombination, and the genomic variability it confers cannot be the sole function of the dimeric genome, since retroviruses with highly conserved genomes and little sequence variability such as HTLV-1 [24] are also dimeric. Translation and packaging? Another possible role is that of the dimer linkage acting as a switch, its presence permitting or restricting the packaging of RNA. In HIV-2 two regions were originally suggested as dimer initiation sites, one analogous to the palindromic sequence identified as the principal DIS in HIV-1 (termed SL1), one close to the PBS [25-27]. Recently, a region upstream of SL1 (also called the DIS, see Figure 1a) was identified as being critical for packaging [28]. An extensive deletion analysis of the 5' leader of HIV-2 was carried out, and removal of nucleotides 380–404 (HIVROD), termed the DM region, rendered the virus severely packaging deficient. The mutation had been designed based on the mfold [29,30] prediction, that removal of these sequences would disrupt the SL1 structure and hence dimerisation (Figure 1b). In vitro studies using RNA transcripts comprising the leader region with and without the DM deletion, reveal that it does, indeed, render the viral RNA monomeric (personal observations). Using antisense oligonucleotides, another group have demonstrated that this region may, in fact, play a role in the dimerisation process itself [31]. By free energy minimisation this region is predicted to be unstructured, so it is not clear how the RNAs would interact with one another. In addition, whilst the SL1/DIS sequence is conserved amongst HIV-2 and SIV sequences in the database, that within the DM region is less so, and the substitutions which exist would affect the auto-complementarity of the sequence. Figure 1 Structure of the HIV-2 leader region. 1a. Secondary structure model of the HIV-2 leader region based on mfold predictions. Indicated are the putative dimer linkage sites (in red). Also highlighted is the DM region defined as being critical for packaging [28], in blue). 1b. The effect of the DM deletion on the SL1/DIS stem loop. The stem is truncated and the internal bulge altered in approximately half the predicted structures. Figure 2 Structure of the key elements involved in HIV-1 RNA dimerisation. 2a. Secondary structure model of the packaging signal of HIV-1Lai ([64] [65]), containing the principal DLS. 2b Proposed sequence of the RNA dimerisation process in HIV-1Lai. The initial kissing hairpin interaction (including loop B) followed by formation of the extended duplex ([1]). 2c. Loop B, one of the critical elements in the dimer interaction. The flexibility of this internal loop allows the duplex to form ([44]). Figure 3 Dimer linkages of the retroviridae (excluding the lentiviruses). 3a. Loose and tight dimers ([51]). 3b. Imperfect repeats ([66]). 3c. Palindromes ([38]). 3d. GACG loops ([23]). 3e. CAG tri-loops (see Figure 4). Figure 4 Proposed tertiary structure of the HTLV-1 dimer linkage. 4a Stereoview of 3D molecular modelling of a potential structure of the HTLV-1 DIS from nucleotide A730 to A744 using JUMNA ([61]). 4b. Close up of the terminal loop. Bases are coloured as follows: adenine, grey; cytosine, yellow; guanine, orange; and uracil, cyan. One of the key differences between HIV-1 and HIV-2 replication is their modes of packaging [32]. Whilst the Gag protein of the former captures the genomic RNA in trans, the latter uses predominantly a cis mechanism. One might postulate therefore, that, if retroviruses must package a dimeric genome, it is critical in the case of HIV-2 that the genome is dimeric before interacting with the Gag polyprotein. Hence, the effect of mutations in the DM region may be to render the RNA monomeric and thus to severely impair packaging. It is attractive to speculate that the reason packaging itself is not affected by DIS mutations to the same degree in HIV-1 [33] is this difference in protein:RNA interaction. If the RNAs can interact at points other than the principal DIS over time, then perhaps the trans mechanism is less dependent on a high affinity dimer linkage? Particle maturation and viral infectivity A recurring observation amongst investigators is the fact that mutation or deletion of dimer linkage sites causes viral infectivity to be decreased [33]. One explanation for this might be that a dimeric genome is a prerequisite for maturation of virus particles. Certainly, immature HIV-1 particles are non-infectious, and viruses with their DLS mutated have been demonstrated to form only immature particles [34]. The DLS of Human T Cell Lymphotropic Virus Type 1 (HTLV-1) was identified as a 14-nucleotide sequence just downstream of the splice donor [35]. Removal of this region from the leader sequence rendered the RNA monomeric in vitro [24,36]. When this deletion was introduced into the wildtype genome sequence, the only viral replication defect that was observed, following transfection and subsequent infection, was that of impaired infectivity [37]. Likewise, a similar effect was observed when the DLS of Human Foamy Virus was mutated [38]. Parent et al showed that if the RNA of Rous Sarcoma Virus (RSV) was engineered so that it was monomeric, the virus was non-infectious [39]. Interestingly, this group suggested that it might be a difference in localisation of structural proteins and RNA affecting subsequent dimer formation and viral infectivity [40]. This is an area that has not been explored to any extent. Also working with RSV, Bieth and colleagues found that, in an in vitro system, dimer formation appeared to inhibit synthesis of the Gag polyprotein precursor [41]. Structure of the dimer linkage Undoubtedly the best defined dimerisation structure is that involved in the dimer linkage of HIV-1. The discovery of the sequences involved, the subsequent description of the RNA:RNA interaction, and the elucidation of the tertiary interaction are described elsewhere [1]. The initial interaction between the two RNAs appears to be a kissing loop interaction (similar to that seen in the regulation of plasmid replication, [42]) followed by annealing of the two RNAs into an extended duplex (Figure 2b). The sequences contained within the palindrome are remarkably conserved. Using an in vitro selection system it has been possible to demonstrate that the DIS has evolved to satisfy both constraints for optimal dimerisation affinity, and the potential to homodimerise [43]. The dimer linkage is found at the terminal end of Stem Loop 1 (SL1) within the packaging signal region of HIV-1 (Figure 2a). The tertiary structure of the whole SL1 RNA has been determined [44,45] and the structures have helped to determine exactly how the RNAs interact with one another. A number of elements appear to be critical for the dimer interaction: flanking purines and central nucleotides in the palindromic sequence [46] and loop B [47-49]. The tertiary structure of the latter has been described (Figure 2c), and there is some debate as to how flexible this internal loop might be. However, work by Borer et al, examining the interaction of NC with elements of the packaging signal, of which loop B is one, showed that, in fact, both structures might exist, the flexible one allowing NC binding at high affinity [50]. There are similar linkages in other retroviruses. The Avian Leukosis Viruses also interact firstly in a kissing hairpin manner, and then form an extended duplex (Figure 3a, [51]). Palindromes remain a theme throughout many of the viruses investigated to-date. As already mentioned, the DIS of HIV-2 is less well defined than that of HIV-1. Whilst there is a palindromic sequence at the top of a stem loop structure that closely resembles the HIV-1 DIS (see Figure 1), there are other regions which have also been demonstrated to be important for dimer formation [25,26]. Other viruses with palindromic sequences as their DLS include HFV (Figure 3c) and MoMLV. In the case of MLV there are other sequences and structures which may play a role in dimer formation, including the GACG tetraloops mentioned previously [52]. The tertiary structure of this stem loop is the only proposed dimer linkage element yet to be determined in a retrovirus other than HIV-1 ([53]). RSV and VL30, also have imperfect repeat sequences in their dimer linkages [54,55] (Figure 3b). Recent work by Monie and colleagues [36] describes the potential tertiary structure of the HTLV-1 dimer linkage, capped by a novel CAG tri-loop (Figure 3e and Figure 4). This tri-loop is formed by an unusual C:synG base pair closing the loop. Other similar loops have been described, in the domain IIId terminal loop of the hepatitis C virus internal ribosomal entry site (IRES) [56] and in stem loops required for initiation of transcription within the Bromoviridae [57]. Although sequence heterogeneity between HTLV-1 isolates is rare, distinct mutations identifying individual strains can be identified. Of 101 HTLV-1 sequences identified from the EMBL database, 90 showed sequence homology with HTLV-1CH, the strain used in the study. The other 11 sequences comprised three different variants. Eight contained a deletion of C736 (see Figure 4), two possessed the substitution A737G, and one possessed the substitution C733U. The substitution mutants have minimal impact on regional secondary structure, while the deletion may induce formation of a CAGG tetraloop. Interestingly, the A737G mutation possesses homology with 150 deposited HTLV-II sequences, suggesting a conservation of the DIS between HTLV-I and -II. Conclusions The retroviral RNA genome structure does not stay static during the course of transcription, translation and ultimately packaging. Various investigators have suggested that this constantly changing RNA structure plays an intimate role in the viral replication [58-61]. It seems possible that linkage of the two RNA molecules constituting the genome is integral to the changes in RNA structure. As described in the article above, the dimer also acts as a mechanism for promoting recombination; may be a signal for packaging to occur; may be an inhibitory signal; may direct processes to occur in specific cellular compartments; and lastly, may be capable of interacting with cellular proteins. In vivo data has revealed just how important an intact dimmer linkage may be to a retrovirus. For instance, there are intriguing differences in the effect of dimer mutations on viral infectivity depending on the cell type being infected [62]. What the significance of this might be in the context of a viral infection is, as yet, unclear. The importance of the dimer linkage is perhaps most clearly exemplified by the observation that a patient infected with a viral isolate having a defective DLS, had a low viral load. The subsequent switch in the predominant virus to that with a competent DLS coincided with a rise in viral load [63]. One can speculate that, at least in the case of HIV-1, only those viruses with a whole, optimised dimer linkage are capable of efficient infectivity. For the purposes of examining the role of retroviral RNA dimer sequences in the context of animal models, the non-human retroviruses, including the non-primate lentiviruses will be of great importance. To sum up, retroviral dimeric genomes are linked by a variety of RNA structures, including kissing hairpins, GACG tetraloops and unusual CAG-tri loops. The differences in these interactions, and when or where they occur, may reflect different demands upon this unique feature, and highlight the elasticity of the RNA genome. Competing interests None declared. Acknowledgements Many thanks to Andrew Lever for advice on, and editing of the manuscript. Particular thanks also go to Tom Monie for Figure 4, allowing description of unpublished work, and critical reading of the manuscript. In addition, thanks to Martin Zaccharias and Jose Gallego for contributions to the other figures, Paul Digard and Ian Brierley for critique. ==== Refs Paillart JC Shehu-Xhilaga M Marquet R Mak J Dimerization of retroviral RNA genomes:an inseparable pair Nat Rev Microbiol 2004 2 461 472 15152202 10.1038/nrmicro903 Russell RS Liang C Wainberg MA Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, No, Probably? Retrovirology 2004 1 23 15345057 Ellermann V Bang O Experimentelle Leukamie bei Huhnern Zentralbl Bakteriol Parasitenkd Infectionskr Hyg Abt Orig 1908 595 609 Rous P A sarcoma of the fowl transmissable by an agent separable from the tumor cells J Ex Med 1911 13 397 411 10.1084/jem.13.4.397 Duesberg PH Physical properties of Rous Sarcoma Virus RNA Proc Natl Acad Sci U S A 1968 60 1511 1518 4299948 Robinson WS Robinson HL Duesberg PH Tumor virus RNA's Proc Natl Acad Sci U S A 1968 58 825 834 4293266 Kung HJ Hu S Bender W Bailey JM Davidson N Nicolson MO McAllister RM RD-114, baboon, and woolly monkey viral RNA's compared in size and structure Cell 1976 7 609 620 182377 10.1016/0092-8674(76)90211-7 Kurg A Sommer G Metspalu A An RNA stem-loop structure involved in the packaging of bovine leukemia virus genomic RNA in vivo Virology 1995 211 434 442 7645247 10.1006/viro.1995.1425 Bender W Chien YH Chattopadhyay S Vogt PK Gardner MB Davidson N High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures J Virol 1978 25 888 896 205678 Bender W Davidson N Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncovirus RNA molecules Cell 1976 7 595 607 182376 10.1016/0092-8674(76)90210-5 Hoglund S Ohagen A Goncalves J Panganiban AT Gabuzda D Ultrastructure of HIV-1 genomic RNA Virology 1997 233 271 279 9217051 10.1006/viro.1997.8585 Brahic M Vigne R Properties of Visna Virus Particles Harvested at Short Time Intervals: RNA Content, Infectivity, and Ultrastructure J Virol 1975 15 1222 1230 16789155 Darlix JL Gabus C Nugeyre MT Clavel F Barre-Sinoussi F Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1 J Mol Biol 1990 216 689 699 2124274 Balakrishnan M Fay PJ Bambara RA The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region J Biol Chem 2001 276 36482 36492 11432862 10.1074/jbc.M102860200 Balakrishnan M Roques BP Fay PJ Bambara RA Template dimerization promotes an acceptor invasion-induced transfer mechanism during human immunodeficiency virus type 1 minus-strand synthesis J Virol 2003 77 4710 4721 12663778 10.1128/JVI.77.8.4710-4721.2003 Andersen ES Jeeninga RE Damgaard CK Berkhout B Kjems J Dimerization and template switching in the 5' untranslated region between various subtypes of human immunodeficiency virus type 1 J Virol 2003 77 3020 3030 12584327 10.1128/JVI.77.5.3020-3030.2003 St Louis DC Gotte D Sanders-Buell E Ritchey DW Salminen MO Carr JK McCutchan FE Infectious molecular clones with the nonhomologous dimer initiation sequences found in different subtypes of human immunodeficiency virus type 1 can recombine and initiate a spreading infection in vitro J Virol 1998 72 3991 3998 9557686 Berkhout B Das AT van Wamel JL The native structure of the human immunodeficiency virus type 1 RNA genome is required for the first strand transfer of reverse transcription Virology 1998 249 211 218 9791013 10.1006/viro.1998.9321 Mougel M Tounekti N Darlix JL Paoletti J Ehresmann B Ehresmann C Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization Nucleic Acids Res 1993 21 4677 4684 8233816 Fisher J Goff SP Mutational analysis of stem-loops in the RNA packaging signal of the Moloney murine leukemia virus Virology 1998 244 133 145 9581786 10.1006/viro.1998.9090 Ly H Nierlich DP Olsen JC Kaplan AH Functional characterization of the dimer linkage structure RNA of moloney murine sarcoma virus [In Process Citation] J Virol 2000 74 9937 9945 11024121 10.1128/JVI.74.21.9937-9945.2000 De Tapia M Metzler V Mougel M Ehresmann B Ehresmann C Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278–303) and new roles for stem-loops H2 (310–352) and H3 (355–374) Biochemistry 1998 37 6077 6085 9558346 10.1021/bi9800303 Rasmussen SV Mikkelsen JG Pedersen FS Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences J Mol Biol 2002 323 613 628 12419254 10.1016/S0022-2836(02)00966-X Greatorex JS Laisse V Dockhelar MC Lever AM Sequences involved in the dimerisation of human T cell leukaemia virus type-1 RNA Nucleic Acids Res 1996 24 2919 2923 8760874 10.1093/nar/24.15.2919 Dirac AM Huthoff H Kjems J Berkhout B The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome J Biol Chem 2001 276 32345 32352 11397802 10.1074/jbc.M103462200 Jossinet F Lodmell JS Ehresmann C Ehresmann B Marquet R Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1 J Biol Chem 2001 276 5598 5604 11092889 10.1074/jbc.M008642200 Lanchy JM Lodmell JS Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro J Mol Biol 2002 319 637 648 12054860 10.1016/S0022-2836(02)00369-8 Griffin SD Allen JF Lever AM The major human immunodeficiency virus type 2 (HIV-2) packaging signal is present on all HIV-2 RNA species: cotranslational RNA encapsidation and limitation of Gag protein confer specificity J Virol 2001 75 12058 12069 11711596 10.1128/JVI.75.24.12058-12069.2001 Zuker M On finding all suboptimal foldings of an RNA molecule Science 1989 244 48 52 2468181 Zuker M Mathews DH Turner DH Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide: NATO ASI Series 1999 Kluwer Academic Publishers Lanchy JM Rentz CA Ivanovitch JD Lodmell JS Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro Biochemistry 2003 42 2634 2642 12614158 10.1021/bi0271190 Kaye JF Lever AM Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation J Virol 1999 73 3023 3031 10074152 Greatorex J Lever A Retroviral RNA dimer linkage J Gen Virol 1998 79 2877 2882 9880000 Liang C Rong L Cherry E Kleiman L Laughrea M Wainberg MA Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins J Virol 1999 73 6147 6151 10364374 Monie T Greatorex J Lever AM Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1 Virus Res 2001 78 45 56 11520579 10.1016/S0168-1702(01)00283-0 Monie TP Greatorex JS Zacharias M Lever AM The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs Biochemistry 2004 43 6085 6090 15147192 10.1021/bi030237i Le Blanc I Greatorex J Dokhelar MC Lever AM A 37 base sequence in the leader region of human T-cell leukaemia virus type I is a high affinity dimerization site but is not essential for virus replication J Gen Virol 2000 81 Pt 1 105 108 10640547 Cain D Erlwein O Grigg A Russell RA McClure MO Palindromic sequence plays a critical role in human foamy virus dimerization J Virol 2001 75 3731 3739 11264362 10.1128/JVI.75.8.3731-3739.2001 Parent LJ Cairns TM Albert JA Wilson CB Wills JW Craven RC RNA dimerization defect in a Rous sarcoma virus matrix mutant J Virol 2000 74 164 172 10590103 Garbitt RA Albert JA Kessler MD Parent LJ trans-Acting Inhibition of Genomic RNA Dimerization by Rous Sarcoma Virus Matrix Mutants J Virol 2001 75 260 268 11119596 10.1128/JVI.75.1.260-268.2001 Bieth E Gabus C Darlix JL A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro Nucleic Acids Res 1990 18 119 127 2155394 Persson C Wagner EGH Nordstrom N Control of replication of plasmid R1: structures and sequences of the antisense RNA, CopA, required for its binding to the target RNA, CopT J Mol Biol 1990 9 3767 3775 Lodmell JS Ehresmann C Ehresmann B Marquet R Convergence of natural and artificial evolution on an RNA loop-loop interaction: the HIV-1 dimerization initiation site RNA 2000 6 1267 1276 10999604 10.1017/S1355838200000698 Greatorex J Gallego J Varani G Lever A Structure and stability of wild-type and mutant RNA internal loops from the SL-1 domain of the HIV-1 packaging signal J Mol Biol 2002 322 543 557 12225748 10.1016/S0022-2836(02)00776-3 Lawrence DC Stover CC Noznitsky J Wu Z Summers MF Structure of the intact stem and bulge of HIV-1 Psi-RNA stem-loop SL1 J Mol Biol 2003 326 529 542 12559920 10.1016/S0022-2836(02)01305-0 Paillart JC Berthoux L Ottmann M Darlix JL Marquet R Ehresmann B Ehresmann C A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis J Virol 1996 70 8348 8354 8970954 Takahashi K Baba S Hayashi Y Koyanagi Y Yamamoto N Takaku H Kawai G NMR analysis of intra- and inter-molecular stems in the dimerization initiation site of the HIV-1 genome J Biochem (Tokyo) 2000 127 681 686 10739962 Takahashi KI Baba S Chattopadhyay P Koyanagi Y Yamamoto N Takaku H Kawai G Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome RNA 2000 6 96 102 10668802 10.1017/S1355838200991635 Shen N Jette L Wainberg MA Laughrea M Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization J Virol 2001 75 10543 10549 11581429 10.1128/JVI.75.21.10543-10549.2001 Yuan Y Kerwood DJ Paoletti AC Shubsda MF Borer PN Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 × 3 internal loop Biochemistry 2003 42 5259 5269 12731867 10.1021/bi034084a Polge E Darlix JL Paoletti J Fosse P Characterization of loose and tight dimer forms of avian leukosis virus RNA J Mol Biol 2000 300 41 56 10864497 10.1006/jmbi.2000.3832 D'Souza V Melamed J Habib D Pullen K Wallace K Summers MF Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition J Mol Biol 2001 314 217 232 11718556 10.1006/jmbi.2001.5139 Kim CH Tinoco I Jr A retroviral RNA kissing complex containing only two Gmiddle dotC base pairs [In Process Citation] Proc Natl Acad Sci U S A 2000 97 9396 9401 10931958 10.1073/pnas.170283697 Torrent C Gabus C Darlix JL A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer J Virol 1994 68 661 667 8289369 Lear AL Haddrick M Heaphy S A study of the dimerization of Rous sarcoma virus RNA in vitro and in vivo Virology 1995 212 47 57 7676649 10.1006/viro.1995.1452 Klinck R Westhof E Walker S Afshar M Collier A Abol-Ela F A potential RNA drug target in the hepatitis C virus internal ribosomal entry site RNA 2000 6 1423 1431 11073218 10.1017/S1355838200000935 Kim CH Kao CC Tinoco I Jr RNA motifs that determine specificity between a viral replicase and its promoter Nat Struct Biol 2000 7 415 423 10802741 10.1038/75202 Huthoff H Berkhout B Two alternating structures of the HIV-1 leader RNA RNA 2001 7 143 157 11214176 10.1017/S1355838201001881 Huthoff H Berkhout B Multiple secondary structure rearrangements during HIV-1 RNA dimerization Biochemistry 2002 41 10439 10445 12173930 10.1021/bi025993n Abbink TE Berkhout B A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon J Biol Chem 2003 278 11601 11611 12458192 10.1074/jbc.M210291200 Andersen ES Contera SA Knudsen B Damgaard CK Besenbacher F Kjems J Role of the Trans-activation Response Element in Dimerization of HIV-1 RNA J Biol Chem 2004 279 22243 22249 15014074 10.1074/jbc.M314326200 Hill MK Shehu-Xhilaga M Campbell SM Poumbourios P Crowe SM Mak J The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells J Virol 2003 77 8329 8335 12857902 10.1128/JVI.77.15.8329-8335.2003 Huthoff H Das AT Vink M Klaver B Zorgdrager F Cornelissen M Berkhout B A human immunodeficiency virus type 1-infected individual with low viral load harbors a virus variant that exhibits an in vitro RNA dimerization defect J Virol 2004 78 4907 4913 15078972 10.1128/JVI.78.9.4907-4913.2004 Harrison GP Lever AM The human immunodeficiency virus type 1 packaging signal and major splice donor region have a conserved stable secondary structure J Virol 1992 66 4144 4153 1602537 Harrison GP Miele G Hunter E Lever AM Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line J Virol 1998 72 5886 5896 9621050 Torrent C Bordet T Darlix JL Analytical study of rat retrotransposon VL30 RNA dimerization in vitro and packaging in murine leukemia virus J Mol Biol 1994 240 434 444 8046749 10.1006/jmbi.1994.1459

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==== Front RetrovirologyRetrovirology1742-4690BioMed Central London 1742-4690-1-231534505710.1186/1742-4690-1-23ReviewIs HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably? Russell Rodney S [email protected] Chen [email protected] Mark A [email protected] McGill AIDS Centre, Lady Davis Institute, Jewish General Hospital, 3755 Cote Ste-Catherine Road Montreal, Quebec, Canada H3T 1E22 Department of Microbiology & Immunology Montreal, Quebec, Canada H3A 2B43 Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 2B42004 2 9 2004 1 23 23 15 7 2004 2 9 2004 Copyright © 2004 Russell et al; licensee BioMed Central Ltd.2004Russell et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets. ==== Body Introduction The dimeric feature of the retroviral RNA genome was identified almost forty years ago. However, as with many topics in retrovirology, interest in this area was heightened with the realization that the causative agent of AIDS was a retrovirus. Since then, RNA and protein sequences involved in genome dimerization have been identified for a number of retroviruses, and the dimeric nature of the retroviral genome is known to be important for various critical events in the viral life cycle. These include reverse transcription and recombination, as well as genome encapsidation. To date, a number of informative reviews have been published on retroviral RNA dimerization [1-3], genome packaging [3-7], and the role of nucleocapsid (NC) protein in these activities [8,9]. More recently, a comprehensive review was published that summarized the contributions of in vitro analysis to the identification of retroviral dimerization signals, and provided an overview of the HIV-1 5' untranslated region (UTR) structure with reference to a number of proposed models [10]. Another, in this issue of Retrovirology, focuses on the different roles of different dimer linkage structures amongst various retroviruses [11]. In this review, we will focus on results from in vivo studies that provide insights into the relationship between retroviral RNA dimerization and packaging, and the biological relevance of these activities to viral replication. Retroviral RNA dimerization The first evidence for the existence of a dimerized RNA genome came in 1967 when it was shown that viral RNA from each of Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), murine leukemia virus (MLV), and mouse mammary tumor virus (MTV) displayed sedimentation constants between 64S and 74S in sucrose gradients [12]. Since these sedimentation constants and corresponding molecular weights were much larger than those of most other known viral RNAs, the structure of these RNA genomes became a matter of great interest. Experiments showing that the 62S RSV RNA species could be converted to a 36S species by heat treatment suggested a disaggregation of the 62S RNA into smaller RNAs, and implied that the fast-sedimenting (62S) RSV RNA was actually an aggregate of smaller (36S) RNAs [13]. The first real understanding of this putative aggregate RNA structure came in 1975 when RNA from the endogenous feline retrovirus, RD-114, was visualized by electron microscopy (EM), and it was apparent that the 52S RNA molecule existed as an extended single strand that contained a central Y- or T-shaped secondary structure [14]. It appeared that this 52S molecule actually consisted of two half-size molecules, joined together by the Y- or T-shaped structure, which was termed rabbit ears (RE). It was later shown that the RNA had a poly(A) sequence at each of the two free ends. More importantly, this indicated that nucleotides involved in this RE, or dimer linkage structure (DLS), resided in the 5' region of the RNA genome [15]. Similar structures were also reported for numerous other type C RNA viruses [16-21]. The absolute conservation of a DLS among retroviruses was strong evidence that the dimerization process must be critical to the retroviral life cycle. With the discovery that the causative agent of AIDS was also a retrovirus, inhibition of RNA dimerization was proposed as a possible therapy for HIV, and HIV-1 RNA dimerization became an intensely studied topic. Both in vivo and in vitro approaches have been used to study retroviral RNA dimerization. The in vivo approach is that whereby RNA is isolated from virions produced in tissue culture and then analyzed by native Northern blotting [22]. The other method involves synthesis of short segments of viral RNA in vitro, and then studying the ability of these fragments to form dimers. The HIV-1 DLS was originally identified when it was shown that an in vitro-transcribed fragment of HIV-1 RNA could form two major bands on a native gel after incubation at 37°C for 15 min [23]. The lower band had the expected size of the RNA fragment, while the upper band corresponded to a dimer. In vivo evidence for a role of the NC protein in the dimerization process was already available [24], and this study also showed that NC could bind to viral RNA and increase the rate of dimerization of the RNA fragments in these in vitro dimerization assays [25]. It was subsequently reported that an RNA fragment representing nt 1–311 of HIV-1 RNA (Mal strain; a chimera of subtypes A and D) could not only form dimers, but that RNAs containing these first 311 nt could dimerize 10 times faster than RNA sequences at positions 311–415 that were previously shown to be sufficient for HIV-1 RNA dimerization [25]. Based on these results, the authors concluded that sequences upstream of the splice donor site are involved in the dimerization process, and proposed that sequences in this region somehow hastened the reaction. The key nucleotides involved in this RNA dimerization event make up a palindromic sequence, 274-GUGCAC-279, between the PBS and the major splice donor [26], and RNA sequences on both sides of this palindrome can form a stem-loop structure with the palindrome in the hairpin loop. Deletion of this stem-loop motif (nt 265–287) completely abolished dimerization of the 1–615 HIV-1 RNA fragment in vitro. The palindromic region was termed the dimerization initiation site (DIS) and it was proposed that this structural element could be exploited for targeted antiviral therapy by antisense oligonucleotides [26]. These findings were later confirmed when a 19 nt sequence upstream of the 5' major SD was shown to be part of the HIV-1 RNA dimerization domain (Lai strain; subtype B) [27], and it was found that in vitro dimerization of a 224–402 nt RNA fragment was completely blocked by an antisense oligonucleotide that targeted the palindrome [28]. This led to a "loop-loop kissing complex" [29] or "kissing-loop model" [27] of HIV-1 RNA dimerization, in which the 6 nt palindromes on each of the two monomeric RNA molecules interact through Watson-Crick base-pairing. Purine residues flanking the palindrome were later shown to be intricately involved in this initial interaction [30,31] which is believed to shift the equilibrium toward the formation of dimers, allowing the stems to melt and anneal to their complementary sequences on the other RNA molecule, thus forming the stable extended duplex (Fig. 1). This model fits with the idea that immature virions contain a less stable dimer involving only base-pairing of the palindromes, but that the mature virions contain a more stable structure, the extended duplex. Subsequent phylogenetic analysis of over 50 HIV-1, HIV-2, and simian immunodeficiency virus (SIV) nucleotide sequences showed an absolute conservation of a predictable structure similar to the DIS, with the hallmark of the HIV-1 DIS motif being a 6 nt palindrome consisting of either a GCGCGC or a GUGCAC sequence [32,33]. Similar kissing-loop models have also been proposed for a number of other retroviruses [34-41]. Figure 1 HIV-1 5' RNA Structural Elements. Illustration of a working model of the HIV-1 5' UTR showing the various stem-loop structures important for virus replication. These are the TAR element, the poly(A) hairpin, the U5-PBS complex, and stem-loops 1–4 containing the DIS, the major splice donor, the major packaging signal, and the gag start codon, respectively. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Clever et al. [73] and Berkhout and van Wamel [136]) Despite ample in vitro evidence supporting the above model of dimer maturation, it was not yet known where or when the RNA dimer was actually formed in vivo. However, native Northern blotting analysis of RNA from two Moloney murine leukemia virus (MuLV) protease-negative (PR-) mutants displayed dimers that migrated more slowly, and showed lower melting temperatures, than that of wild-type [42]. It was therefore concluded that PR function is required for RNA maturation in MuLV. Similar experiments with a related virus also suggested that the RNA maturation event required an intact, unsubstituted Cys array within the NC domain [42]. On the basis of these results, a maturation pathway was proposed for MuLV in which Gag polyprotein molecules assemble into a nascent virion containing an immature dimer. The particle would then be released from the cell, and once Gag is cleaved by PR, NC would act on the immature dimer, converting it to the mature form. Evidence for the role of NC in this dimer maturation process came when in vitro analysis showed that NC could convert the less thermostable dimers to a more stable conformation [43]. Similar results were obtained by others showing that HIV-1 NC could activate dimerization of a 77–402 nt fragment of HIV-1 Lai RNA, as well as convert an unstable dimer, corresponding to the kissing complex, to a stable one [44]. Taken together, these thermostability conversions seem to resemble the RNA maturations reported in vivo, and, in agreement with earlier proposals [24], strongly suggest that NC is responsible for the dimer maturation depicted in Fig. 1. Subsequent in vivo analysis of a panel of HIV-1 NC mutants showed that Cys-Ser substitution of amino acid residues within the second zinc finger decreased genomic RNA dimerization to the same extent as disruption of the DIS [45]. This finding confirmed the involvement of NC in the dimerization process, and suggests that the kissing-loop model also applies to the in vivo situation. HIV-1 RNA packaging Why a class of viruses would evolve to have such a unique genomic structure is not entirely clear, but it is speculated that the availability of two copies of the genome would be advantageous for recombination during the complex reverse transcription process, that is key to the retroviral life cycle [46]. Indeed, the dimeric nature of the genome is thought to be responsible for a high rate of recombination during infection [47-50]. Given that most dimerization signals overlap with known packaging elements, it was naturally assumed that it is the RNA dimer that is specifically recognized for packaging in the case of retroviruses, and that this dimeric feature ensures proper packaging of two copies of genomic RNA. A number of studies have attempted to address this question of a link between dimerization and packaging, but let us first review several aspects of the HIV-1 RNA packaging process. The first studies aimed at identifying the HIV-1 RNA packaging signal found that deletion of RNA sequences between the major splice donor (SD) and the gag coding region (i.e. SL3 and adjacent sequences in Fig. 2) decreased the levels of genomic RNA packaged into virions [51-53]. Since these sequences were downstream of the major 5' SD, and therefore would not be found in any spliced viral RNA species, it was plausible that this region could be responsible for the selective packaging of genomic RNAs. Analysis of the putative ψ locus from a variety of retroviruses showed that these sequences had the ability to direct the selective encapsidation of heterologous RNAs to which they had been linked artificially [54-61]. In HIV-1, such autonomous packaging signals were mapped to the regions extending 30–40 nt immediately upstream and downstream of the gag start codon [62]; however, subsequent studies showed that RNA sequences upstream of the 5' SD site also affected RNA packaging [63]. It was also known that retroviral encapsidation required trans-acting amino acid sequences in the Gag protein [51,64-68], and several groups reported that HIV-1 Gag and NC exhibit specific binding affinity for the HIV-1 ψ site in vitro [23,69-72]. These findings, combined with chemical and RNase accessibility mapping, as well as computerized sequence analysis, led to the generation of a model for the HIV-1 ψ site that comprised four independent stem-loops [73] (SL1-4 in Fig. 2). Three of these hypothetical stem-loop structures were each shown to serve as independent Gag binding sites, and were proposed to contribute individually to overall packaging efficiency. SL1, SL3, and SL4 were later shown to be critical for packaging specificity in vivo [74,75]. Subsequent in vitro analysis from another group demonstrated that the major packaging signal is an extended bulged stem-loop whose RNA conformation is altered upon interaction with Gag [76]. However, more recent work indicates that SL2 and SL3 display much higher affinities for NC than SL1 and SL4 in vitro [77,78]. Based on these findings, a model has been proposed to represent the initial complex formed between the NC domains of assembling Gag molecules and the dimeric ψ region [79]. In this model, SL1 is shown to form an RNA duplex between the two stands, while SL4, instead of directly binding to Gag, contributes additional RNA-RNA interactions that stabilize the tertiary structure of the ψ element. The RNA conformation resulting from this folding pattern is thought to expose SL2 and SL3 for high-affinity binding to Gag. Figure 2 The Kissing-Loop Model of HIV-1 RNA Dimerization. HIV-1 RNA dimerization is initiated by a Watson-Crick base-pairing interaction between two palindromes in the loops of SL1 on two monomeric genomic RNAs. This interaction forms the loose unstable kissing-loop complex. Coincident with virus particle maturation, this unstable dimer is rearranged to form a more stable extended duplex that involves a mechanism whereby the base-pairs in the stems melt and then re-anneal to their complementary sequences on the opposite strand. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Skripkin et al. [26] and Laughrea and Jetté [27]) Despite the clear results obtained from simplified in vitro studies such as those mentioned above, the SL1-4 region alone is not sufficient to target RNA into HIV-1 virions in vivo [80], and the minimal region required to confer autonomous packaging activity actually maps to a larger region covering the first 350–400 nt of the genome, including ≈ 240 nt upstream of SL1 [81-84]. In agreement with these studies, mutations that alter the stability of the poly(A) hairpin stem region, or delete the upper part of the hairpin, severely inhibited HIV-1 replication [85]. And, these deficits in replication correlated with reduced RNA packaging levels in virions, suggesting that the formation of the poly(A) hairpin is necessary for normal packaging of viral genomes. Subsequent research confirmed the importance of the poly(A) hairpin in the RNA packaging process [86], and it was shown that similar disruption of base-pairing in the stem of the TAR element also caused profound defects in packaging [81,86]. Finally, deletion analyses of RNA sequences between the poly(A) hairpin and SL1 suggested that unspecified sequences within the U5-PBS region also contribute to HIV-1 RNA packaging [83,86]. Our group later showed that GU-rich sequences in the lower stems of the poly(A) hairpin and the U5-PBS complex contribute to both dimerization and packaging [87]. In summary, all of the seven predicted stem-loop structures in the HIV-1 5' UTR (Fig. 2) are known to be important for genome encapsidation, and all of these RNA structural elements have also been assigned other functions in various steps of the viral life cycle, e.g. the role of SL1 in the initiation of dimerization. The existence of such overlapping functions for these RNA structures raises the possibility that some of these functions, such as dimerization and packaging, might be linked. The evidence for and against the existence of such a link in HIV-1 will be the main focus of the remainder of this review. Is dimerization a prerequisite for packaging? One of the first electron microscopy studies of a retroviral DLS in 1976 proposed that this region "could have some role in packaging the RNA in the virus" [16]. This raised the question of a possible link between dimerization and packaging that is still debated. The answer to this question has significance in our basic understanding of the retroviral life cycle and may also have implications for therapy, since many groups are actively studying these two activities as potential drug targets. Clues from in vitro studies Early reports on in vitro dimerization of HIV-1 RNA showed that the DLS localized to a stretch of genomic RNA downstream of the 5' SD (nt 311–415) [23,88], and it was noted that this dimerization domain encompassed a previously identified packaging element that had also been shown to bind NC [51-53]. This dependence of HIV-1 RNA dimerization on cis elements required for packaging was immediately interpreted to mean that retroviral RNA dimerization, activated by either NC or Gag precursors, should direct genomic RNA into the virion, implying that dimerization might be a prerequisite for packaging. Since HIV-1, MuLV, and RSV all contain elements involved in dimerization that were also required for packaging [23,89,90], it was proposed that dimerization might function as a molecular switch that negatively regulates translation and positively regulates encapsidation [88]. The existence of a DLS downstream of the major splice donor would seemingly supply a convenient mechanism whereby only genome length RNA would be able to dimerize and subsequently become encapsidated into the virion. However, evidence questioning such a dimerization-mediated mechanism of genomic RNA packaging came from studies showing that sequences upstream of the SD site had even greater dimerization capabilities than those located downstream [25-27]. The involvement of such sequences (e.g. the DIS, SL1) in the dimerization process questioned the link between dimerization and packaging, because these sequences are also found in all HIV-1 spliced viral RNAs. Observations from in vivo studies Early in vivo studies analyzing the structure of virion-associated RNA from rapid-harvest avian retroviruses showed that viral RNA appeared to be a mixture of monomers and dimers [91-93]. Similar results had also been reported with PR [94,95] and NC [24,94,96,97] mutants, which argued against the notion that dimerization is a prerequisite for packaging. However, analysis of rapid-harvest virus in MuLV showed that genomic RNA was already in the form of a dimer shortly after budding, albeit as a less stable, physically different RNA dimer than that present in mature virions [42]. Based on these observations it was proposed that MuLV particles never package monomeric RNAs, but rather that the dimeric RNA structure might be integral to the packaging signal that is recognized by Gag during assembly. It was also speculated that the previously reported presence of monomers in viral RNA preparations had resulted from the physical dissociation of fragile unstable dimers during RNA preparation. Similar experiments performed on PR- mutants of HIV-1 showed that substantial amounts of monomeric RNA could be detected [98]. Since PR- dimers were shown to be less stable than wild-type dimers, it was assumed that dimers were preferentially packaged in PR- particles, but that some fragile dimeric structures had dissociated during RNA preparation. Based on these in vivo results with both MuLV and HIV-1, it was concluded that dimerization is a prerequisite for packaging and should be considered to be a general feature of retrovirus assembly. Further insights into this topic can be obtained by examination of results from a number of studies aimed at understanding the role of the DIS in HIV-1 replication. One such study, in which DIS loop palindrome sequences were mutated, found that mutation of the palindrome to shorter or longer versions of GC stretches did not have major effects on viral RNA dimerization; however, partial RNA packaging defects were observed that also corresponded to diminutions in viral replication [33]. Based on these data, it was proposed that these DIS loop mutants might have experienced a partial dimerization defect that caused inefficient packaging [33]. In a similar study, mutation of the palindrome, as well as deletion of the upper stem-loop of SL1 caused drastic reductions in viral infectivity and decreases in both dimerization and packaging of HIV-1 genomic RNA [32]. In an attempt to explain how these mutations could affect both activities, a model was proposed in which Gag does not specifically recognize the dimerized genome but rather initially interacts with one molecule of genomic RNA that happens to be linked (dimerized) to a second such molecule. Then, during packaging, Gag would effectively bind to two genomic RNA molecules at once. Hence, defects in dimerization would result in subsequent packaging defects. Based on these data, it was also concluded that the encapsidation and dimerization processes are coupled to some extent. Although several groups had attempted to delineate the relationship between dimerization and packaging, the fact remains that the RNA signals that are important for both of these activities overlap in most retroviral genomes; this makes it difficult to interpret the results of mutagenesis studies. In an attempt to generate viruses that would be expected to display selective defects in dimerization or packaging, one group designed a panel of constructs containing mutations in SL1, SL3, or both [99]. Results from this study showed that deletion of either SL1 alone, or SL3 plus adjacent flanking sequences, reduced genomic packaging, while deletion of SL1 and SL3 simultaneously caused an even further reduction. With respect to dimerization, complete deletion of SL1, or even disruption of the base-pairing in the upper stem, resulted in elevated levels of monomer-sized RNA species on native Northern blots, again confirming the importance of this region for the in vivo HIV-1 RNA dimerization process. Yet, these mutant genomes could still be packaged, suggesting that HIV-1 RNAs need not be dimers for this to happen. Thus, the authors concluded that dimerization is not a prerequisite for packaging but rather serves an independent function in the retroviral life cycle. In the above-cited article, the effects of SL3 mutations on dimerization were not studied, but our group later showed that viruses containing even minor substitutions in or around SL3 could have significant effects on both dimerization and packaging [100,101]. In summary, the in vivo studies described above commonly observed that mutations in 5' RNA sequences affected both dimerization and packaging, presumably due to the close proximity of the RNA dimerization and packaging signals. Can monomers be packaged? In an attempt to separate the dimerization and packaging functions, and to characterize the DIS-DLS region without altering packaging activity, one group generated mutant constructs carrying a duplication of approximately 1000 nt from the HIV-1 5' region (termed E/DLS) including the encapsidation signal and the DIS-DLS [102]. They found that the presence of an ectopic E/DLS near the 3' region of the genome resulted in the appearance of monomeric RNA in virus particles, suggesting that monomers can be packaged and that dimerization of HIV-1 genomic RNA is not required for packaging. However, they also found that two intact E/DLS regions had to be present on the same RNA molecule in order for packaging of monomers to occur. Therefore, it was assumed that these monomers had been generated from an intramolecular interaction between the two E/DLS regions. If we assume that such an intramolecular interaction between two DLS structures would occur on a single RNA molecule, however, might such a structure then not also appear as a dimer to a Gag protein that was attempting to package it? Although these data were interpreted to mean that dimerization is not required for packaging, they also suggest that some structure that is generated by the interaction of the two E/DLS regions might be recognized by Gag in order to facilitate packaging. In the context of wild-type genomic RNA containing only one E/DLS region, such a structure might then only be generated by an intermolecular interaction between two RNA molecules, i.e. a dimer. Hence, these results also imply that dimerization might be required for proper packaging. In a follow-up study, the same group created mutant HIV-1 particles that contained only monomeric RNAs, and concluded that these mutants demonstrated the complete separation of encapsidation from physical dimerization of retroviral RNA [103]. However, they also reported that these viruses packaged only monomers, and that packaging efficiencies were approximately half those of wild-type, implying that dimerization is the sole mechanism to ensure the packaging of two copies of viral genomic RNA into each virus particle. In addition, the packaged monomers might have originally been weak dimers that dissociated during extraction and analysis, as has been pointed out in previous reports [42,102]. However, the above results do raise the issue of packaging specificity in mutant viruses. We and others have shown that, in COS cells, HIV-1 can incorporate significant amounts of spliced viral RNA when proper packaging of full-length viral genomic RNA is reduced [99,104]. During assembly, Gag will always successfully package some RNA, and it is important to know the degree of specificity with which monomers versus dimers are packaged. If monomer-packaging mutants concomitantly package high levels of spliced viral RNA, then it is likely that packaging specificity may have been compromised by the existence of an extra E/DLS, and that the packaging of the monomers was non-specific. However, a lack of spliced viral RNA in these virions would indicate that the monomers were packaged with a high degree of specificity, and would have implications as to whether or not Gag initially recognizes viral genomic RNA in a dimeric versus a monomeric state. None of the viruses engineered to package only monomers were able to efficiently establish a new round of infection, suggesting that dimerization is required for replication if not for packaging. It is difficult to predict what other effects the addition of large segments of highly structured RNA might have on the viral life cycle. Another group reported similar phenotypes in the context of an HIV-1 mutant that was designed to have altered Gag/Gag-Pol ratios [105]. Analysis of virion-derived genomic RNA from these viruses showed an increase in packaging of monomers, demonstrating that stable RNA dimers are not required for encapsidation of HIV-1 genomic RNA. Interestingly, these viruses also showed drastically reduced infectivity. Insights from forced evolution studies We have also been studying the HIV-1 5' UTR and its putative interactions with Gag, and how these interactions affect dimerization and packaging activities. The DIS is known to be important for viral replication [32,33,63,99,106-109], reverse transcription [47,48,107,109], RNA dimerization [32,99,106,109-111], and packaging [32,33,74,99,107,108,110], as well as packaging specificity [99]. However, despite the obvious importance of this stem-loop structure, work from our group has shown that defective viral replication caused by deletions in the DIS can be largely corrected by a series of compensatory point mutations identified in matrix, capsid, p2, and NC [112-114]. These findings imply that the RNA sequences comprising the DIS interact in some way with these domains of Gag, and that when the RNA sequences are mutated, the virus will acquire adaptive mutations that potentially restore putative RNA-protein interactions over long-term culture. Since the originally deleted RNA sequences were in the DIS, we had naturally assumed that the major defect of these mutants would relate to RNA dimerization, and that compensatory mutations had arisen to correct defective RNA dimerization activity. To our surprise, this was not the case. Although our mutants did indeed yield reduced levels of dimerized genomic RNA in virus particles, the compensatory mutations in Gag that restored replication capacity [112-114] did not correct dimerization defects [109]. Rather, compensatory mutations apparently resulted in increased overall levels of viral genomic RNA that were packaged into virus particles, irrespective of impaired RNA dimerization. Similar effects on packaging were observed in the context of compensatory mutations identified during long-term culture of viruses containing mutations outside the DIS, such as the poly(A) hairpin and the U5-PBS complex [87], and between the PBS and SL1 [115]. These findings again question the link between dimerization and packaging, since our compensatory point mutations were able to increase RNA packaging levels without correcting dimerization. One possibility is that the revertant viruses somehow gained the ability to package wild-type levels of RNA without correcting dimerization defects, i.e. they packaged more monomers. However, we also cannot rule out the possibility that our point mutations in Gag may have restored weak dimerization properties to the mutated RNAs, and that the latter dimers dissociated during extraction and analysis. In a follow-up study, we created two other DIS deletions and combined them with various combinations of the previously identified compensatory point mutations. We showed that these mutant viruses, ΔLoop (lacking the loop region of SL1) and ΔDIS (lacking the complete SL1) displayed defects in replication, RNA dimerization, and packaging. Once more, all of these but dimerization were largely corrected by the compensatory point mutations in Gag [104]. Even a virus that lacked the DIS, e.g. ΔDIS, and which never showed any signs of viral growth in tissue culture, was able to replicate to significant extent when it also possessed the compensatory mutations. The mechanism(s) whereby these compensatory point mutations functioned to restore replication had eluded us for some time. Recently, however, we employed an RNase protection assay to discriminate between genomic and spliced viral RNA packaged into virus particles. Our results showed that all of our 5' UTR mutant viruses aberrantly packaged increased levels of spliced viral RNA compared to wild-type virions. More importantly, however, the effect of one of our compensatory point mutations (i.e. MP2; a Thr->Ile substitution at position 12 of the SP1 spacer peptide in Gag) was to exclude spliced viral RNA from being packaged into mutant virions [104]. Surprisingly, this single point mutation was also able to restore significant levels of virus replication to our ΔDIS mutant virus, which had been noninfectious in both T cell lines and blood mononuclear cells. Previous work had suggested that the packaging of spliced viral RNA is a mechanism used by packaging mutants to fill the space that would normally be occupied by genomic RNA [99]. Were this the case, then the MP2-mediated exclusion of spliced viral RNA from the virus particle should have been accompanied by increased packaging of genomic RNA. In the absence of MP2, the mutant particles contained lower levels of genomic RNA and higher levels of spliced viral RNA packaged than wild-type. In contrast, the presence of MP2 led to the exclusion of spliced viral RNA, but had no effect on packaging of genomic RNA. In the context of dimerization and packaging in the mutated viruses, it is possible that spliced viral RNAs, which do contain some RNA elements involved in RNA dimerization, including the DIS, might form heterodimers with molecules of genomic RNA. These putative heterodimers might be packageable, but it is unlikely that virions containing such genomes would be able to replicate, e.g. the noninfectious ΔDIS mutant. However, in the presence of MP2, the modified Gag protein might in some way block the formation of such an RNA heterodimer, thereby increasing the probability that dimers form between two genomic RNA molecules, resulting in partially restored levels of virus replication. Since these genomic RNA molecules are already mutated in dimerization signals, these weaker dimers would probably appear on a gel as monomers. In such a model, MP2 would act to restore dimerization, resulting in increased replication capacity, suggesting that dimerization is required for proper packaging to ensure that a particle is infectious. Unfortunately, this is virtually impossible to prove with current in vitro and in vivo protocols. New approaches to study dimerization and packaging within the cell will hopefully allow new hypotheses to be tested. The packaging of spliced viral RNA and/or the exclusion of such RNA species raises the question of whether the viral RNA sequence, or possibly the RNA structure, is important in proper assembly and/or structural integrity of the virus particle itself. Evidence in support of this possibility comes from studies on the binding of NC, in the context of full-length Gag, to viral genomic RNA. This might concentrate Gag proteins onto one or more RNA molecules, thereby facilitating Gag-Gag multimerization in a template-driven manner. Hence, viral genomic RNA would be a structural element, or scaffold, on which the virion can assemble [116]. Other reports have shown that viral RNA can affect particle morphogenesis [116-119] and structural stability [120,121], although the mechanisms involved are unclear. If RNA structure, or even the dimeric versus monomeric state of the RNA, truly does play a role in virion assembly and/or stability, this might also explain the apparent detection of monomeric RNA in the HIV-1 mutants mentioned above. For example, the duplication of large E/DLS sequences would undoubtedly have altered the overall structure of viral RNA, which might have resulted in the formation of unstable virus particles [102,103]. Degradation of such particles could have indirectly caused the dissociation of dimers that would then appear as monomers on a gel. The fact that these viruses were all noninfectious may also have been due to the formation of unstable virus particles. Consistent with this concept, we found by electron microscopy that HIV-1 mutants lacking DIS stem sequences displayed an increased proportion of immature virus particles [114]. This might mean that either the RNA structure, or the lack of a properly formed dimer, resulted in the production of virus particles with abnormal morphology. Since RNA can affect Gag cleavage, it is possible that mutations in the RNA might have also compromised the cleavage of Gag precursor proteins, which may subsequently have affected particle maturation [122]. We believe that proper RNA dimerization may be a prerequisite for efficient virion assembly and structural stability. As stated, the link between dimerization and packaging is a subject of ongoing debate [32,33,42,98,99,102,103,109,110], but we and others view dimerization as a prerequisite for packaging. Genomic RNA can be packaged as monomers [99,102,103,105,109], or alternatively as weak dimers that appear as monomers on gels, but mutant viruses that exhibit dimerization defects generally do not grow as well as wild-type viruses. The fact that our ΔDIS-MP2 virus can replicate in tissue culture, despite being severely compromised in genome dimerization, is evidence that efficient dimerization is not required for packaging or replication. In the absence of an authentic DIS, other sequences that affect dimerization may form a weak dimer that allows RNA to be recognized and adequately packaged [87,100,101]. The contribution of the DIS might then be to significantly increase the efficiency of the dimerization process, resulting in more efficient packaging and replication. In conclusion, we agree with opinions expressed by others that the generation of virus particles able to package monomeric genomes is possible, but that dimerization is likely to be a prerequisite for the production of infectious viral progeny [10]. The DIS as a therapeutic target? It is clear that virus replication capacity is significantly affected whenever dimerization and/or packaging are compromised, suggesting that these activities can be exploited as anti-HIV drug targets. Indeed, the DIS was first proposed to be a potential therapeutic target at least 10 years ago, and antisense molecules were directed at this region of viral RNA [26,123], without practical outcome. Other approaches directly target the HIV-1 kissing-loop complex, which resembles the eubacterial 16S ribosomal aminoacyl-tRNA site, i.e. the target of aminoglycoside antibiotics such as paramycin and neomycin [124], both of which specifically bind to the kissing-loop complex. Drugs based on antibiotics with high affinity and specificity for the DIS may be a worthwhile approach, although efficacy might be compromised by the fact that HIV can replicate in the face of mutations that decrease genomic dimerization by more than 50% [104]. RNA interference (RNAi) is a novel mechanism that regulates gene expression in which small interfering RNAs direct the targeted degradation of RNA in a sequence-specific manner (reviewed in Lee and Rossi [125]). Although RNAi is a powerful tool, it is not yet clear whether its therapeutic potential will materialize. This not-withstanding, several reports show that specific degradation of HIV-1 RNA is possible in infected cells [125], and reductions of p24 levels by as much as 4 logs have been achieved using RNAi directed against HIV-1 tat and rev [126]. DNA vectors are currently being engineered that will allow for long-term production of siRNAs for use against chronic diseases, such as HIV-1. The DIS might also be a good candidate for sequence-specific targeting of HIV by RNAi therapy since it is highly conserved among naturally occurring virus isolates, and, due to its position upstream of the major splice donor, is contained in all HIV-1 RNA transcripts, both spliced and unspliced. Effective DIS-directed degradation of HIV RNA should confer the same viral phenotype as observed with our ΔDIS mutant, which never showed signs of virus replication in either permissive T cell lines or blood mononuclear cells [104]. One concern with use of RNAi is how accessible certain RNA sequences might be. For example, complex secondary structures might cause some sequences to be buried and therefore inaccessible to the siRNA. However, this would not be a concern with DIS-directed RNAi, since the DIS contains a 6 nt palindromic sequence that is believed to initiate the dimerization process by binding to an identical sequence on another molecule of genomic RNA. If two 6 nt stretches of RNA can find each other on two 9200 nt strands of highly structured RNA, they should also be accessible to siRNAs. Recently, the practicality of RNAi-based therapies against HIV-1 was called into question when it was shown that HIV-1 was able to escape the antiviral pressure of RNAi by generating substitutions or even deletions within RNAi target sequences [127,128]. This again highlights the versatility and plasticity of the HIV-1 genome. However, in these studies, the RNAi target sequences were located within the tat and nef genes, and the mutations that were generated blocked the effects of the RNAi without conferring any major detriment to virus replication. In contrast, RNAi may be more useful if targeted to more critical RNA elements within the genome, such as the DIS or the Ψ region, since any escape mutations that occur might result in viruses with severely impaired replication ability. All of these DIS-directed strategies rely on specifically targeting the viral RNA itself, which might not be practical given our inadequate knowledge of the overall structure of the HIV-1 5' region. The fact that RNA sequences such as SL1 and SL3 are known to form relevant RNA-protein interactions raises the possibility that the protein component of these interactions might also provide potential targets for anti-HIV therapy. Such approaches are currently being explored in research aimed at designing inhibitors of the TAR-Tat RNA-protein interaction [129]. Similar approaches might also be developed to target RNA-protein interactions involving SL1 or SL3 and Gag. Future directions Current HIV combination therapies have demonstrated that a multi-targeted approach against the virus results in the greatest degree of suppression of virus replication. Therefore, the identification of novel targets for anti-HIV therapy could significantly improve HIV treatment strategies. HIV-1 RNA dimerization is clearly a critical event that could be exploited as a target once its complete mechanism is elucidated. It is pleasing to see that a number of laboratories that have actively researched RNA dimerization and packaging are now moving beyond conventional in vitro and in vivo approaches toward more biologically relevant methods. One group has taken chemical modification protocols commonly used for in vitro RNA analysis, and adapted them for use in virus-producing cells. Hence, structural analysis of viral RNA, that would previously be carried out only in vitro on short fragments of artificially transcribed RNA, can now be performed on in vivo-generated HIV-1 genomic RNA (J.-C. Paillart and R. Marquet, personal communication, and [130]). This method also allows comparisons of cellular and virion-derived HIV-1 RNA and represents a middle ground between classic in vitro and in vivo approaches. The goal of this work is to provide insight on the true structure of the HIV-1 leader, and on which RNA substructures are involved in dimerization. Preliminary data suggest that viral RNA may already be dimerized in the cytoplasm (J-C. Paillart and R. Marquet, unpublished data). This method might also have application in regard to in vivo foot-printing that could allow the study of RNA-protein interactions in the context of virus-producing cells. The structure of the viral RNA that exists in the cell has long been a topic of interest, and recent data suggest that different RNA sequences might be involved in higher order intrastrand structures that favor the dimerization of the two RNA molecules. Such a model has been proposed [131], and is supported by numerous in vitro dimerization studies conducted on HIV-1, HIV-2, and SIV RNA [41,131-133]. The model proposes that the HIV-1 5' UTR can form two alternating conformations, termed the long-distance interaction (LDI) and the branched multiple hairpin (BMH) structures. The LDI conformation is believed to exist when the RNA is in a monomer form, and is thought to form a long extended base-paired structure with almost all of the proposed stem-loop sequences buried. This structure is thought to be favored during certain steps of the life cycle, such as translation. In this model, NC has been shown to bind the LDI structure to induce a switch to the BMH structure [131], in which the DIS and ψ would then be exposed in a manner able to mediate dimerization and packaging. Such a 'riboswitch' is an attractive hypothesis, especially since similar mechanisms have recently been proposed to account for previously unexplained results in the field of gene regulation [134]. Although there is currently little in vivo evidence directly supporting such a model in the case of retroviruses, the results of previous mutagenesis studies from several laboratories correlate with those that would be predicted from the riboswitch model, both concerning RNA packaging and RNA dimerization status [135]. In regard to dimerization being a prerequisite for packaging, it would also be interesting to test whether an HIV-1 RNA molecule in the LDI conformation can be packaged. Since the BMH conformation is believed to mediate dimerization, one would assume that the LDI structures would not be packageable if dimerization is truly a packaging prerequisite. Others have developed a fluorescence resonance energy transfer (FRET)-based system to allow visualization of RNA-Gag interactions within cells (A.M. Lever and co-workers, unpublished data). Such a system might provide insight into the timing of genome selection and packaging. It will also be interesting to determine whether this system can be adapted to pinpoint how retroviral RNA dimerization takes place within cells, and whether dimerization indeed occurs before RNA is selected for packaging. Competing interests None declared. Author's contributions RSR gathered the information discussed in this review, and was primary author of the manuscript. CL and MAW carefully read the manuscript and offered insightful suggestions for its revision. All authors read and approved the final version. Acknowledgements The authors wish to acknowledge past and present members of the Liang and Wainberg laboratories, for continued contribution to this field. We apologize to those researchers whose work has not been cited due to publication restraints. RSR is the recipient of a Doctoral Research Award from the Canadian Institutes of Health Research (CIHR). CL is a Chercheur-Boursier of the Fonds de la Recherche en Sante du Quebec (FRSQ) and a New Investigator of the CIHR. Research in our labs has been supported by grants from the CIHR, the FRSQ, and the Canadian Foundation for Innovation. We are also grateful to Diane and Aldo Bensadoun for support of our research program. ==== Refs Greatorex J Lever A Retroviral RNA dimer linkage J Gen Virol 1998 79 2877 2882 9880000 Paillart JC Marquet R Skripkin E Ehresmann C Ehresmann B Dimerization of retroviral genomic RNAs: structural and functional implications Biochimie 1996 78 639 653 8955907 10.1016/S0300-9084(96)80010-1 Berkhout B Structure and function of the human immunodeficiency virus leader RNA Prog Nucleic Acid Res Mol Biol 1996 54 1 34 8768071 Lever AM Richardson JH Harrison GP Retroviral RNA packaging Biochem Soc Trans 1991 19 963 966 1794592 Lever AM HIV RNA packaging and lentivirus-based vectors Adv Pharmacol 2000 48 1 28 10987087 10.1016/S1054-3589(00)48002-6 Berkowitz R Fisher J Goff SP RNA packaging Curr Top Microbiol Immunol 1996 214 177 218 8791728 Rein A Retroviral RNA packaging: a review Arch Virol Suppl 1994 9 513 522 8032280 Rein A Henderson LE Levin JG Nucleic-acid-chaperone activity of retroviral nucleocapsid proteins: significance for viral replication Trends Biochem Sci 1998 23 297 301 9757830 10.1016/S0968-0004(98)01256-0 Katz RA Jentoft JE What is the role of the cys-his motif in retroviral nucleocapsid (NC) proteins? Bioessays 1989 11 176 181 2695083 Paillart JC Shehu-Xhilaga M Marquet R Mak J Dimerization of retroviral RNA genomes: an inseparable pair Nat Rev Microbiol 2004 2 461 472 15152202 10.1038/nrmicro903 Greatorex J The retroviral RNA dimer linkage: different structures may reflect diiferent roles Retrovirology 2004 1 22 15317659 10.1186/1742-4690-1-22 Robinson WS Robinson HL Duesberg PH Tumor virus RNA's Proc Natl Acad Sci U S A 1967 58 825 834 4293266 Duesberg PH Physical properties of Rous Sarcoma Virus RNA Proc Natl Acad Sci U S A 1968 60 1511 1518 4299948 Kung HJ Bailey JM Davidson N Nicolson MO McAllister RM Structure, subunit composition, and molecular weight of RD-114 RNA J Virol 1975 16 397 411 168408 Bender W Davidson N Mapping of poly(A) sequences in the electron microscope reveals unusual structure of type C oncornavirus RNA molecules Cell 1976 7 595 607 182376 10.1016/0092-8674(76)90210-5 Kung HJ Hu S Bender W Bailey JM Davidson N Nicolson MO McAllister RM RD-114, baboon, and woolly monkey viral RNA's compared in size and structure Cell 1976 7 609 620 182377 10.1016/0092-8674(76)90211-7 Dube S Kung HJ Bender W Davidson N Ostertag W Size, subunit composition, and secondary structure of the Friend virus genome J Virol 1976 20 264 272 978793 Maisel J Bender W Hu S Duesberg PH Davidson N Structure of 50 to 70S RNA from Moloney sarcoma viruses J Virol 1978 25 384 394 202749 Bender W Chien YH Chattopadhyay S Vogt PK Gardner MB Davidson N High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures J Virol 1978 25 888 896 205678 Gonda MA Rice NR Gilden RV Avian reticuloendotheliosis virus: characterization of the high-molecular-weight viral RNA in transforming and helper virus populations J Virol 1980 34 743 751 6247509 Murti KG Bondurant M Tereba A Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy J Virol 1981 37 411 419 6260992 Khandjian EW Meric C A procedure for Northern blot analysis of native RNA Anal Biochem 1986 159 227 232 2433961 Darlix JL Gabus C Nugeyre MT Clavel F Barre-Sinoussi F Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1 J Mol Biol 1990 216 689 699 2124274 Meric C Spahr PF Rous sarcoma virus nucleic acid-binding protein p12 is necessary for viral 70S RNA dimer formation and packaging J Virol 1986 60 450 459 2430109 Marquet R Paillart JC Skripkin E Ehresmann C Ehresmann B Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site Nucleic Acids Res 1994 22 145 151 8121797 Skripkin E Paillart JC Marquet R Ehresmann B Ehresmann C Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro Proc Natl Acad Sci U S A 1994 91 4945 4949 8197162 Laughrea M Jette L A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA Biochemistry 1994 33 13464 13474 7947755 Muriaux D Girard PM Bonnet-Mathoniere B Paoletti J Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5' leader region is evidenced by an antisense oligonucleotide J Biol Chem 1995 270 8209 8216 7713927 10.1074/jbc.270.14.8209 Paillart JC Skripkin E Ehresmann B Ehresmann C Marquet R A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA Proc Natl Acad Sci U S A 1996 93 5572 5577 8643617 10.1073/pnas.93.11.5572 Ennifar E Walter P Ehresmann B Ehresmann C Dumas P Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site Nat Struct Biol 2001 8 1064 1068 11702070 10.1038/nsb727 Mujeeb A Parslow TG Zarrinpar A Das C James TL NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA FEBS Lett 1999 458 387 392 10570946 10.1016/S0014-5793(99)01183-7 Laughrea M Jette L Mak J Kleiman L Liang C Wainberg MA Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization J Virol 1997 71 3397 3406 9094609 Berkhout B van Wamel JL Role of the DIS hairpin in replication of human immunodeficiency virus type 1 J Virol 1996 70 6723 6732 8794309 Polge E Darlix JL Paoletti J Fosse P Characterization of loose and tight dimer forms of avian leukosis virus RNA J Mol Biol 2000 300 41 56 10864497 10.1006/jmbi.2000.3832 Fosse P Motte N Roumier A Gabus C Muriaux D Darlix JL Paoletti J A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization Biochemistry 1996 35 16601 16609 8987995 10.1021/bi9613786 De Tapia M Metzler V Mougel M Ehresmann B Ehresmann C Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278–303) and new roles for stem-loops H2 (310–352) and H3 (355–374) Biochemistry 1998 37 6077 6085 9558346 10.1021/bi9800303 Girard PM de Rocquigny H Roques BP Paoletti J A model of PSI dimerization: destabilization of the C278-G303 stem-loop by the nucleocapsid protein (NCp10) of MoMuLV Biochemistry 1996 35 8705 8714 8679633 10.1021/bi952454s Girard PM Bonnet-Mathoniere B Muriaux D Paoletti J A short autocomplementary sequence in the 5' leader region is responsible for dimerization of MoMuLV genomic RNA Biochemistry 1995 34 9785 9794 7626648 Tounekti N Mougel M Roy C Marquet R Darlix JL Paoletti J Ehresmann B Ehresmann C Effect of dimerization on the conformation of the encapsidation Psi domain of Moloney murine leukemia virus RNA J Mol Biol 1992 223 205 220 1731069 Jossinet F Lodmell JS Ehresmann C Ehresmann B Marquet R Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1 J Biol Chem 2001 276 5598 5604 11092889 10.1074/jbc.M008642200 Dirac AM Huthoff H Kjems J Berkhout B The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome J Biol Chem 2001 276 32345 32352 11397802 10.1074/jbc.M103462200 Fu W Rein A Maturation of dimeric viral RNA of Moloney murine leukemia virus J Virol 1993 67 5443 5449 8350405 Feng YX Copeland TD Henderson LE Gorelick RJ Bosche WJ Levin JG Rein A HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro Proc Natl Acad Sci U S A 1996 93 7577 7581 8755517 10.1073/pnas.93.15.7577 Muriaux D De Rocquigny H Roques BP Paoletti J NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer J Biol Chem 1996 271 33686 33692 8969239 10.1074/jbc.271.52.33686 Laughrea M Shen N Jette L Darlix JL Kleiman L Wainberg MA Role of distal zinc finger of nucleocapsid protein in genomic RNA dimerization of human immunodeficiency virus type 1; no role for the palindrome crowning the R-U5 hairpin Virology 2001 281 109 116 11222101 10.1006/viro.2000.0778 Hu WS Temin HM Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination Proc Natl Acad Sci U S A 1990 87 1556 1560 2304918 Andersen ES Jeeninga RE Damgaard CK Berkhout B Kjems J Dimerization and template switching in the 5' untranslated region between various subtypes of human immunodeficiency virus type 1 J Virol 2003 77 3020 3030 12584327 10.1128/JVI.77.5.3020-3030.2003 Balakrishnan M Fay PJ Bambara RA The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region J Biol Chem 2001 276 36482 36492 11432862 10.1074/jbc.M102860200 Balakrishnan M Roques BP Fay PJ Bambara RA Template dimerization promotes an acceptor invasion-induced transfer mechanism during human immunodeficiency virus type 1 minus-strand synthesis J Virol 2003 77 4710 4721 12663778 10.1128/JVI.77.8.4710-4721.2003 van Wamel JL Berkhout B The first strand transfer during HIV-1 reverse transcription can occur either intramolecularly or intermolecularly Virology 1998 244 245 251 9601495 10.1006/viro.1998.9096 Aldovini A Young RA Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus J Virol 1990 64 1920 1926 2109098 Clavel F Orenstein JM A mutant of human immunodeficiency virus with reduced RNA packaging and abnormal particle morphology J Virol 1990 64 5230 5234 2204725 Lever A Gottlinger H Haseltine W Sodroski J Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions J Virol 1989 63 4085 4087 2760989 Adam MA Miller AD Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions J Virol 1988 62 3802 3806 3418786 Armentano D Yu SF Kantoff PW von Ruden T Anderson WF Gilboa E Effect of internal viral sequences on the utility of retroviral vectors J Virol 1987 61 1647 1650 3033290 Bender MA Palmer TD Gelinas RE Miller AD Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region J Virol 1987 61 1639 1646 3502707 Embretson JE Temin HM Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line J Virol 1987 61 2675 2683 3039161 Knight JB Si ZH Stoltzfus CM A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA J Virol 1994 68 4493 4502 8207823 Mann R Baltimore D Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs J Virol 1985 54 401 407 3989912 Sullenger BA Cech TR Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA Science 1993 262 1566 1569 8248806 Watanabe S Temin HM Encapsidation sequences for spleen necrosis virus, an avian retrovirus, are between the 5' long terminal repeat and the start of the gag gene Proc Natl Acad Sci U S A 1982 79 5986 5990 6310558 Hayashi T Shioda T Iwakura Y Shibuta H RNA packaging signal of human immunodeficiency virus type 1 Virology 1992 188 590 599 1585635 10.1016/0042-6822(92)90513-O Kim HJ Lee K O'Rear JJ A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA Virology 1994 198 336 340 8259668 10.1006/viro.1994.1037 Dorfman T Luban J Goff SP Haseltine WA Gottlinger HG Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein J Virol 1993 67 6159 6169 8371356 Gorelick RJ Nigida SM JrBess JW JrArthur LO Henderson LE Rein A Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA J Virol 1990 64 3207 3211 2191147 Morellet N Jullian N De Rocquigny H Maigret B Darlix JL Roques BP Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR Embo J 1992 11 3059 3065 1639074 Omichinski JG Clore GM Sakaguchi K Appella E Gronenborn AM Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy FEBS Lett 1991 292 25 30 1959614 10.1016/0014-5793(91)80825-N South TL Summers MF Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC Protein Sci 1993 2 3 19 8443588 Sakaguchi K Zambrano N Baldwin ET Shapiro BA Erickson JW Omichinski JG Clore GM Gronenborn AM Appella E Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein Proc Natl Acad Sci U S A 1993 90 5219 5223 8506369 Luban J Goff SP Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA J Virol 1994 68 3784 3793 8189516 Dannull J Surovoy A Jung G Moelling K Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues Embo J 1994 13 1525 1533 8156990 Berkowitz RD Luban J Goff SP Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays J Virol 1993 67 7190 7200 8230441 Clever J Sassetti C Parslow TG RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1 J Virol 1995 69 2101 2109 7884856 McBride MS Panganiban AT The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures J Virol 1996 70 2963 2973 8627772 McBride MS Panganiban AT Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo J Virol 1997 71 2050 2058 9032337 Zeffman A Hassard S Varani G Lever A The major HIV-1 packaging signal is an extended bulged stem loop whose structure is altered on interaction with the Gag polyprotein J Mol Biol 2000 297 877 893 10736224 10.1006/jmbi.2000.3611 De Guzman RN Wu ZR Stalling CC Pappalardo L Borer PN Summers MF Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element Science 1998 279 384 388 9430589 10.1126/science.279.5349.384 Amarasinghe GK De Guzman RN Turner RB Chancellor KJ Wu ZR Summers MF NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition J Mol Biol 2000 301 491 511 10926523 10.1006/jmbi.2000.3979 Amarasinghe GK Zhou J Miskimon M Chancellor KJ McDonald JA Matthews AG Miller RR Rouse MD Summers MF Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition J Mol Biol 2001 314 961 970 11743714 10.1006/jmbi.2000.5182 Berkowitz RD Hammarskjold ML Helga-Maria C Rekosh D Goff SP 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal Virology 1995 212 718 723 7571442 10.1006/viro.1995.1530 Helga-Maria C Hammarskjold ML Rekosh D An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA J Virol 1999 73 4127 4135 10196309 Kaye JF Richardson JH Lever AM cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging J Virol 1995 69 6588 6592 7666564 McBride MS Schwartz MD Panganiban AT Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation J Virol 1997 71 4544 4554 9151848 Parolin C Dorfman T Palu G Gottlinger H Sodroski J Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes J Virol 1994 68 3888 3895 7910642 Das AT Klaver B Klasens BI van Wamel JL Berkhout B A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication J Virol 1997 71 2346 2356 9032371 Clever JL Eckstein DA Parslow TG Genetic dissociation of the encapsidation and reverse transcription functions in the 5' R region of human immunodeficiency virus type 1 J Virol 1999 73 101 109 9847312 Russell RS Hu J Laughrea M Wainberg MA Liang C Deficient dimerization of human immunodeficiency virus type 1 RNA caused by mutations of the u5 RNA sequences Virology 2002 303 152 163 12482667 10.1006/viro.2002.1592 Marquet R Baudin F Gabus C Darlix JL Mougel M Ehresmann C Ehresmann B Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism Nucleic Acids Res 1991 19 2349 2357 1645868 Bieth E Gabus C Darlix JL A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro Nucleic Acids Res 1990 18 119 127 2155394 Prats AC Roy C Wang PA Erard M Housset V Gabus C Paoletti C Darlix JL cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA J Virol 1990 64 774 783 2153242 Cheung KS Smith RE Stone MP Joklik WK Comparison of immature (rapid harvest) and mature Rous sarcoma virus particles Virology 1972 50 851 864 4344944 10.1016/0042-6822(72)90439-4 Korb J Travnicek M Riman J The oncornavirus maturation process: quantitative correlation between morphological changes and conversion of genomic virion RNA Intervirology 1976 7 211 224 188781 Canaani E Helm KV Duesberg P Evidence for 30–40S RNA as precursor of the 60–70S RNA of Rous sarcoma virus Proc Natl Acad Sci U S A 1973 70 401 405 4119788 Oertle S Spahr PF Role of the gag polyprotein precursor in packaging and maturation of Rous sarcoma virus genomic RNA J Virol 1990 64 5757 5763 1700822 Stewart L Schatz G Vogt VM Properties of avian retrovirus particles defective in viral protease J Virol 1990 64 5076 5092 1697912 Bowles NE Damay P Spahr PF Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein J Virol 1993 67 623 631 8380458 Dupraz P Oertle S Meric C Damay P Spahr PF Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein J Virol 1990 64 4978 4987 2168981 Fu W Gorelick RJ Rein A Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions J Virol 1994 68 5013 5018 8035501 Clever JL Parslow TG Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation J Virol 1997 71 3407 3414 9094610 Rong L Russell RS Hu J Laughrea M Wainberg MA Liang C Deletion of stem-loop 3 is compensated by second-site mutations within the Gag protein of human immunodeficiency virus type 1 Virology 2003 314 221 228 14517075 10.1016/S0042-6822(03)00405-7 Russell RS Hu J Beriault V Mouland AJ Laughrea M Kleiman L Wainberg MA Liang C Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA J Virol 2003 77 84 96 12477813 10.1128/JVI.77.1.84-96.2003 Sakuragi J Shioda T Panganiban AT Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions J Virol 2001 75 2557 2565 11222678 10.1128/JVI.75.6.2557-2565.2001 Sakuragi J Iwamoto A Shioda T Dissociation of genome dimerization from packaging functions and virion maturation of human immunodeficiency virus type 1 J Virol 2002 76 959 967 11773371 Russell RS Roldan A Detorio M Hu J Wainberg MA Liang C Effects of a single amino acid substitution within the p2 region of human immunodeficiency virus type 1 on packaging of spliced viral RNA J Virol 2003 77 12986 12995 14645555 10.1128/JVI.77.24.12986-12995.2003 Shehu-Xhilaga M Crowe SM Mak J Maintenance of the Gag/Gag-Pol ratio is important for human immunodeficiency virus type 1 RNA dimerization and viral infectivity J Virol 2001 75 1834 1841 11160682 10.1128/JVI.75.4.1834-1841.2001 Laughrea M Shen N Jette L Wainberg MA Variant effects of non-native kissing-loop hairpin palindromes on HIV replication and HIV RNA dimerization: role of stem-loop B in HIV replication and HIV RNA dimerization Biochemistry 1999 38 226 234 9890902 10.1021/bi981728j Paillart JC Berthoux L Ottmann M Darlix JL Marquet R Ehresmann B Ehresmann C A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis J Virol 1996 70 8348 8354 8970954 Harrison GP Miele G Hunter E Lever AM Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line J Virol 1998 72 5886 5896 9621050 Shen N Jette L Liang C Wainberg MA Laughrea M Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging J Virol 2000 74 5729 5735 10823883 10.1128/JVI.74.12.5729-5735.2000 Sakuragi J Ueda S Iwamoto A Shioda T Possible role of dimerization in human immunodeficiency virus type 1 genome RNA packaging J Virol 2003 77 4060 4069 12634365 10.1128/JVI.77.7.4060-4069.2003 Laughrea M Jette L HIV-1 genome dimerization: kissing-loop hairpin dictates whether nucleotides downstream of the 5' splice junction contribute to loose and tight dimerization of human immunodeficiency virus RNA Biochemistry 1997 36 9501 9508 9235995 10.1021/bi970862l Liang C Rong L Laughrea M Kleiman L Wainberg MA Compensatory point mutations in the human immunodeficiency virus type 1 Gag region that are distal from deletion mutations in the dimerization initiation site can restore viral replication J Virol 1998 72 6629 6636 9658109 Liang C Rong L Quan Y Laughrea M Kleiman L Wainberg MA Mutations within four distinct gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site J Virol 1999 73 7014 7020 10400801 Liang C Rong L Cherry E Kleiman L Laughrea M Wainberg MA Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins J Virol 1999 73 6147 6151 10364374 Liang C Rong L Russell RS Wainberg MA Deletion mutagenesis downstream of the 5' long terminal repeat of human immunodeficiency virus type 1 is compensated for by point mutations in both the U5 region and gag gene J Virol 2000 74 6251 6261 10864634 10.1128/JVI.74.14.6251-6261.2000 Muriaux D Mirro J Harvin D Rein A RNA is a structural element in retrovirus particles Proc Natl Acad Sci U S A 2001 98 5246 5251 11320254 10.1073/pnas.091000398 Campbell S Rein A In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain J Virol 1999 73 2270 2279 9971810 Campbell S Vogt VM Self-assembly in vitro of purified CA-NC proteins from Rous sarcoma virus and human immunodeficiency virus type 1 J Virol 1995 69 6487 6497 7666550 Ganser BK Li S Klishko VY Finch JT Sundquist WI Assembly and analysis of conical models for the HIV-1 core Science 1999 283 80 83 9872746 10.1126/science.283.5398.80 Wang SW Aldovini A RNA incorporation is critical for retroviral particle integrity after cell membrane assembly of Gag complexes J Virol 2002 76 11853 11865 12414928 10.1128/JVI.76.23.11853-11865.2002 Wang SW Noonan K Aldovini A Nucleocapsid-RNA interactions are essential to structural stability but not to assembly of retroviruses J Virol 2004 78 716 723 14694103 10.1128/JVI.78.2.716-723.2004 Sheng N Pettit SC Tritch RJ Ozturk DH Rayner MM Swanstrom R Erickson-Viitanen S Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease J Virol 1997 71 5723 5732 9223458 Skripkin E Paillart JC Marquet R Blumenfeld M Ehresmann B Ehresmann C Mechanisms of inhibition of in vitro dimerization of HIV type I RNA by sense and antisense oligonucleotides J Biol Chem 1996 271 28812 28817 8910525 10.1074/jbc.271.46.28812 Ennifar E Paillart JC Marquet R Ehresmann B Ehresmann C Dumas P Walter P HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics J Biol Chem 2003 278 2723 2730 12435744 10.1074/jbc.M205726200 Lee NS Rossi JJ Control of HIV-1 replication by RNA interference Virus Res 2004 102 53 58 15068880 10.1016/j.virusres.2004.01.015 Lee NS Dohjima T Bauer G Li H Li MJ Ehsani A Salvaterra P Rossi J Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells Nat Biotechnol 2002 20 500 505 11981565 Boden D Pusch O Lee F Tucker L Ramratnam B Human immunodeficiency virus type 1 escape from RNA interference J Virol 2003 77 11531 11535 14557638 10.1128/JVI.77.21.11531-11535.2003 Das AT Brummelkamp TR Westerhout EM Vink M Madiredjo M Bernards R Berkhout B Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition J Virol 2004 78 2601 2605 14963165 10.1128/JVI.78.5.2601-2605.2004 Xie B Calabro V Wainberg MA Frankel AD Selection of TAR RNA-binding chameleon peptides by using a retroviral replication system J Virol 2004 78 1456 1463 14722301 10.1128/JVI.78.3.1456-1463.2004 Goldschmidt V Paillart JC Rigourd M Ehresmann B Aubertin AM Ehresmann C Marquet R Structural variability of the initiation complex of HIV-1 reverse transcription J Biol Chem 2004 279 35923 35931 15194685 10.1074/jbc.M404473200 Huthoff H Berkhout B Multiple secondary structure rearrangements during HIV-1 RNA dimerization Biochemistry 2002 41 10439 10445 12173930 10.1021/bi025993n Abbink TE Berkhout B A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon J Biol Chem 2003 278 11601 11611 12458192 10.1074/jbc.M210291200 Huthoff H Berkhout B Two alternating structures of the HIV-1 leader RNA RNA 2001 7 143 157 11214176 10.1017/S1355838201001881 Knight J Gene regulation: switched on to RNA Nature 2003 425 232 233 13679885 10.1038/425232a Ooms M Huthoff H Russell RS Liang C Berkhout B A riboswitch regulates RNA dimerization and packaging in HIV-1 virions J Virol 2004 Berkhout B van Wamel JL The leader of the HIV-1 RNA genome forms a compactly folded tertiary structure RNA 2000 6 282 295 10688366 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==== Front Reprod HealthReproductive Health1742-4755BioMed Central London 1742-4755-1-31535786310.1186/1742-4755-1-3ResearchWHO systematic review of maternal morbidity and mortality: the prevalence of severe acute maternal morbidity (near miss) Say Lale [email protected] Robert C [email protected]ülmezoglu A Metin [email protected] UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction, Department of Reproductive Health and Research, World Health Organization, Geneva, Switzerland2 MRC Maternal and Infant Health Care Strategies Research Unit, University of Pretoria, Kalafong Hospital, South Africa2004 17 8 2004 1 3 3 26 7 2004 17 8 2004 Copyright © 2004 Say et al; licensee BioMed Central Ltd.2004Say et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aim To determine the prevalence of severe acute maternal morbidity (SAMM) worldwide (near miss). Method Systematic review of all available data. The methodology followed a pre-defined protocol, an extensive search strategy of 10 electronic databases as well as other sources. Articles were evaluated according to specified inclusion criteria. Data were extracted using data extraction instrument which collects additional information on the quality of reporting including definitions and identification of cases. Data were entered into a specially constructed database and tabulated using SAS statistical management and analysis software. Results A total of 30 studies are included in the systematic review. Designs are mainly cross-sectional and 24 were conducted in hospital settings, mostly teaching hospitals. Fourteen studies report on a defined SAMM condition while the remainder use a response to an event such as admission to intensive care unit as a proxy for SAMM. Criteria for identification of cases vary widely across studies. Prevalences vary between 0.80% – 8.23% in studies that use disease-specific criteria while the range is 0.38% – 1.09% in the group that use organ-system based criteria and included unselected group of women. Rates are within the range of 0.01% and 2.99% in studies using management-based criteria. It is not possible to pool data together to provide summary estimates or comparisons between different settings due to variations in case-identification criteria. Nevertheless, there seems to be an inverse trend in prevalence with development status of a country. Conclusion There is a clear need to set uniform criteria to classify patients as SAMM. This standardisation could be made for similar settings separately. An organ-system dysfunction/failure approach is the most epidemiologically sound as it is least open to bias, and thus could permit developing summary estimates. ==== Body Background Severe acute maternal morbidity (SAMM), also known as "near miss", is defined as "A very ill pregnant or recently delivered woman who would have died had it not been that luck and good care was on her side" [1,2]. This concept is relatively new in maternal care, but is increasingly becoming important in areas with low maternal mortality ratios or where the geographic area is small [3,4]. The use of data collected on SAMM has been shown to be a mechanism for identifying health system failures or priorities in maternal health care more rapidly than maternal deaths [5]. It has the advantage of events still being rare enough not to overload clinicians and data capturing personnel within a facility. Its routine use as an indicator, however, is limited due to the lack of uniform criteria of identification of the cases. This study was undertaken to systematically review all available studies on SAMM with a view to establishing the global prevalence and examining the usefulness as a maternal health indicator. Methods This study is a part of a bigger systematic review undertaken by the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), Department of Reproductive Health and Research at the World Health Organization (WHO) to obtain prevalence/incidence data on maternal mortality and a range of morbidities including SAMM. The methodology of the systematic review followed an a priori protocol and involved an extensive search of all relevant published/unpublished data from 1997 to 2002. The methodology of the systematic review and the search strategy have been described elsewhere [6]. In brief, we searched 10 electronic databases, WHO regional databases, internet and reference lists, contacted experts in the field, and hand-searched relevant articles in the WHO Library. Criteria for inclusion of studies in the review were: inclusion of data relevant to pre-defined conditions, specified dates for data collection period, including data from 1990 onwards, sample size >200 and a clear description of methodology. A data extraction instrument was used to extract data from included studies. This instrument includes 48 items distributed in five modules three of which were relevant to this analysis. Modules were designed to collect information on (i) the general study level characteristics such as design, population, setting, (ii) prevalence/incidence of maternal morbid conditions, and (iii) quality assessment of morbidity reports. Reporting of definitions and of the procedures used for identification of cases for morbidities were part of quality assessment. We did not assign quality scores to articles, but preferred to present available information on variables regarded as quality components (including reporting of definitions, case-identification criteria, characteristics of setting and participants). Nearly 65 000 reports were screened initially by titles and/or abstracts of which more than 4500 were retrieved for full-text evaluation. More than 2500 of these were included in the review. Data extracted were entered into a specifically constructed database and tabulated using SAS software. A small number of the articles in the review report on SAMM, near miss or a similar definition such as severe morbidity, critically ill obstetric patient. Although we included other articles reporting on individual severe morbidities within their own category of conditions (e.g. severe hypertension within hypertensive disorders of pregnancy), this particular article is concerned with those papers which define a separate entity of SAMM or similar, or those which report on the most recognised end-points for SAMM (i.e. emergency hysterectomy and obstetric admissions to intensive care units). We later conducted an updated search of MEDLINE and Popline using the keywords 'near miss morbidity' and 'severe maternal morbidity' to find out about any recent publications that could be of importance, but were not included in the database due to the end date of the original search (2002) for the bigger systematic review. The date of this complementary search was June 2004. We describe below the included studies with an emphasis on the different definitions used and criteria for identification of the cases. Results A total of 30 reports of SAMM are included in the systematic review (twenty-seven identified by the original search and three by the complementary search). Study designs are mainly cross-sectional and most of them are conducted in tertiary care hospitals (see Additional file 1). Most of the studies describe the characteristics of the setting and participants as well as reporting definitions and procedures for identification of the cases. There are essentially two types of definition of severe acute maternal morbidity; one describing what the authors meant by a near miss; and the other describing a response to an event such as hysterectomy or admission to intensive care unit (ICU). Fourteen studies define a specific SAMM or near-miss 'condition' [1,2,7-18] while nine consider admissions to ICU as near-miss cases [19-27] and the remainder report on emergency hysterectomies [28-34]. In the majority of articles there is an intuitive agreement on what a near miss means – a woman who almost died but survived. Identification of cases, however, is complex and varies widely across studies. We listed the articles in Additional file 1 in three categories according to criteria used to classify patients as being near miss; disease-specific (specified criteria for common conditions, e.g. pre-eclampsia, haemorrhage); management-specific (specified criteria related to response to disease, e.g. hysterectomy or admission to ICU); and organ-system dysfunction/failure based (specified criteria for dysfunction or failure related to each organ system). One study reports the proportion of the admissions to ICU separately as well as the total number of SAMM cases [8]. We included this report under the section of organ-system based identification criteria. Individual prevalence rates for SAMM vary between 0.80% – 8.23% in the first category of studies that use disease-specific criteria. Case-finding criteria differ significantly within this category as well. In addition, some of the studies with similar criteria use mainly clinician's evaluation for identification [15], while others have established threshold levels for the degree of severity of the conditions of concern [7,18]. This is probably due to contextual factors such as the availability of facilities with sufficient diagnostic tools. The range is 0.38% – 1.09% in the group of reports that use organ-system based criteria and include unselected women. Rates are lower (0.01% – 2.99%) and variation is lesser in the category of studies using management-based criteria. An expected finding is the difference between resource-poor and more advantageous settings in the prevalence of SAMM. In resource-poor settings, 4–8% of pregnant women who deliver in the hospitals will experience SAMM when case-identification criteria are based on specific diseases. This rate is around 1% when the organ failure is considered. In more developed country settings, the rates are around 1% with disease-specific and 0.4% with organ-system based criteria, respectively. The results also suggest that the use of organ-system based criteria is more specific in identifying the real SAMM cases. Discussion Due to the wide variation in identification of the cases, it is not possible to pool data and make a summary estimate for SAMM. Because of the variation in case identifications in the three categories of identification criteria as well as variation within each category (e.g. for disease-specific criteria – the use of physician's evaluation versus technology requiring tests) it is difficult to make comparisons as well. Nevertheless, it is evident that the prevalence of SAMM is higher in studies conducted in less developed country settings. Rates seem to be higher also in studies that use disease-specific criteria as compared to those using organ-system based criteria for similar settings. This finding suggests less specificity of disease-specific criteria in identifying real SAMM cases. Although less specific, the use of disease-specific criteria has some advantages; it is easy to interpret, cases can be identified retrospectively, and the quality of care for that particular disease can be assessed [18,35]. However, the approach concentrates on certain diseases, and thus, other problems such as pulmonary embolus, which is an important cause of maternal death in developed countries could be ignored [36]. In addition, definition of conditions may not always be straightforward. For example, the same threshold for severe haemorrhage could have different consequences in women with normal haemoglobin levels or those with severe anaemia. Furthermore, although detailed objective criteria are established for case identification in developed country settings [18], the limited availability of resources in less developed settings may not permit this level of detail. Therefore, identification of cases is likely to be less accurate when the diagnosis depends on clinical estimates [2,15]. Use of management-specific criteria is advantageous in that it is simple to identify the cases, but it depends on many other variables such as the availability of ICU beds, the facilities in an ICU, or differing views about and indications for hysterectomy. Also, the approach does not include all SAMM cases. One study reporting SAMM according to organ-system based criteria and admissions to ICU separately shows that admissions to ICU represent only one third of all SAMM cases [8]. Use of organ-system based criteria allows for identifying all severe morbidities and then investigating the primary cause, thus does not discard any particular condition. It is the most reproducible across similar areas and criteria can be defined according to resources available. High technology requiring laboratory and haemodynamic investigations can be avoided. However, it is the most labour-intensive for identifying cases, hence criteria for inclusion as near miss must be strict. Bias can be introduced if data collection is incomplete and prevalence can be underestimated. Two approaches are used as potential methods of assessing the care SAMM cases receive. "Mortality Index-MI" is defined as the ratio of maternal deaths among the SAMM cases to the sum of maternal deaths and SAMM cases [35,37]. It represents the proportion of women who presents with a SAMM and subsequently dies [37]. Another approach is to calculate the ratio of SAMM to mortality [8,18]. We attempted to calculate the ratio of SAMM to mortality for all studies included under the categories of disease-specific or organ-system based case identification criteria (see Additional file 1). We did not calculate MI because it was not clear from some reports whether reported maternal deaths were identified as SAMM or not prior to death. It is clearly illustrated in the studies that more SAMM cases are likely to die in resource-poor settings than in more developed country settings. For example, the studies conducted in Niger, Benin and Malaysia give the morbidity to mortality ratio as 11–12 [10,15,17] while this is 117–223 in studies conducted in Europe [11,18] in the category where disease-specific criteria are used. The same applies to the category of organ-system based criteria; morbidity: mortality ratio is 5–8 in South Africa [1,14,15] and 49 in Scotland [8]. These findings suggest that an indicator that relates SAMM to maternal deaths could be a useful method in assessing the care SAMM cases receive. However, the definitions and identification of cases should be standardised at least for similar settings and the indicator needs to be clearly defined. Conclusion Considering all complexities in definition and case-identification of SAMM, it is necessary that studies clearly describe their identification criteria for the cases. There is a clear need to set criteria to identify SAMM cases. Use of organ-system based criteria seems to be a more useful approach in identifying cases as variation in defining criteria can be avoided, particularly for similar settings. It would then be easier to establish summary estimates for SAMM prevalence which could serve as a measure of maternal health and quality of care indicator. Competing interests None declared. Authors' contributions AMG and LS coordinated the conduct of the systematic review of maternal morbidity and mortality. RCP and LS outlined the manuscript. LS reviewed the studies and wrote the initial draft. AMG and RCP substantially improved the manuscript. Supplementary Material Additional file 1 Table describing important variables of all studies included in the systematic review Click here for file Acknowledgements We thank Dr Paul FA Van Look for his thorough review of the manuscript and valuable inputs. ==== Refs Mantel GD Buchmann E Rees H Pattinson RC Severe acute maternal morbidity: a pilot study of a definition for a near-miss Br J Obstet Gynaecol 1998 105 985 990 9763050 Prual A Bouvier-Colle M-H De Bernis L Bréart G Severe maternal morbidity from direct obstetric causes in West Africa: incidence and case fatality rates Bull World Health Organ 2000 78 593 602 10859853 Stones W Lim W Al-Azzawi F Kelly M An investigation of maternal morbidity with identification of life-threatening 'near miss' episodes Health Trends 1991 23 13 15 10113878 Hall MH Near misses and severe maternal morbidity In Why mothers die 1997–1999: The confidential enquiries into maternal deaths in the United Kingdom 2001 London: RCOG Press: Department of Health, Welsh Office, Scottish Home and Health Department, Department of Health and Social Sciences, Northern Ireland 323 325 Drife JO Maternal 'near-miss' reports? BMJ 1993 307 1087 1088 8251802 Gülmezoglu AM Say L Betran AP Villar J Piaggio G WHO systematic review of maternal mortality and morbidity: methodological issues and challenges BMC Med Res Methodol 2004 4 16 15236664 10.1186/1471-2288-4-16 Bouvier-Colle M-H Varnoux N Le groupe MOMS-B Mortalité maternelle et morbidité grave dans trois régions françaises: resultats de MOMS, une enquête européenne multicentrique J Gynecol Obstet Biol Reprod 2001 30 2S5 2S9 Brace V Penney G Hall M Quantifying severe maternal morbidity: a Scottish population study BJOG 2004 111 481 484 15104614 10.1111/j.1471-0528.2004.00101.x Cochet L Macdonald AP Pattinson RC Severe acute maternal morbidity and maternal death audit – a rapid diagnostic tool for evaluating maternal care S Afr Med J 2003 93 700 702 14635560 Filippi V Alihonou E Mukantaganda S Graham WJ Ronsmans C Near misses: maternal morbidity and mortality Lancet 1998 351 145 146 9439527 Girard F Burlet G Bayoumeu F Fresson J Bouvier-Colle M-H Boutroy J-L Les complications sévères de la grossess et de l'accouchement: état des lieux en Lorraine dans le cadre de l'enquête européene J Gynecol Obstet Biol Reprod 2001 30 10 17 Kaye D Mirembe F Aziga F Namulema B Maternal mortality and associated near-misses among emergency intrapartum obstetric referrals in Mulago Hospital, Kampala, Uganda East Afr Med J 2003 80 144 149 12762430 Khosla AH Dahiya K Sangwan K Maternal mortality and 'near-miss' in rural north India Int J Gynecol Obstet 2000 68 163 164 10.1016/S0020-7292(99)00185-X Pattinson RC Vandecruys HI Macdonald AP Mantel GD Why do women die during childbirth 2001 Prual A Huguet D Gabin O Rabe G Severe obstetric morbidity of the third trimester, delivery and early puerperium in Niamey (Niger) Afr J Reprod Health 1998 2 10 19 10214424 Sahel A De Brouwere V Lardi M Van Lerberghe W Ronsmans C Filippi V Obstetric catastrophes barely just avoided: near misses in Moroccan hospitals Sante 2001 11 229 235 11861198 Sivalingam N Looi K Clinical experience with management of 'near-miss' cases in obstetrics Med J Malaysia 1999 54 496 503 11072469 Waterstone M Bewley S Wolfe C Incidence and predictors of severe obstetric morbidity: case-control study BMJ 2001 322 1089 1094 11337436 10.1136/bmj.322.7294.1089 Baskett TF Sternadel J Maternal intensive care and near-miss mortality in obstetrics Br J Obstet Gynaecol 1998 105 981 984 9763049 Ben Letaifa D Daouas N Ben Jazia K Slama A Jegham H Maternal emergencies requiring controlled ventilation: epidemiology and prognosis J Gynecol Obstet Biol Reprod 2002 31 256 260 Bewley S Creighton S Near-miss obstetric enquiry J Obstet Gynaecol (Paris) 1997 17 26 29 Bouvier-Colle M-H Ancel PY Varnoux N Salanave B Fernandez H Papiernik E Bréart G Soins intensifs des femmes en état gravido-puerpéral. Charactéristiques des patientes et structures de prise en charge J Gynecol Obstet Biol Reprod 1997 26 47 56 De Souza JPD Duarte G Basile-Filho A Near-miss maternal mortality in devloping countries Eur J Obstet Gynecol Reprod Biol 2002 104 80 12128271 10.1016/S0301-2115(02)00206-3 Loverro G Greco P Vimercati A Nicolardi V Varcaccio-Garofalo G Selvaggi L Maternal complications associated with cesarean section J Perinat Med 2001 29 322 326 11565201 Murphy DJ Charlett P Cohort study of near-miss maternal mortality and subsequent reproductive outcome Eur J Obstet Gynecol Reprod Biol 2002 102 173 178 11950486 10.1016/S0301-2115(01)00320-7 Rodriguez-Iglesias G Calzado JD Riveiro LP Experiencia de 12 años de trabajo la atención de adolescentes obstétricas criticamente enferma en la unidad de cuidados intensivos Rev Cubana Obstet Ginecol 1999 25 141 145 Ryan M Hamilton V Bowen M McKenna P The role of a high-dependency unit in a regional obstetric hospital Anaesthesia 2000 55 1155 1158 11121922 10.1046/j.1365-2044.2000.01627.x Alsayali ARA Baloul AMA Emergency obstetric hysterectomy: 8-year review at Taif Maternity Hospital, Saudi Arabia Ann Saudi Med 2000 20 454 456 17264649 Bakshi S Meyer BA Indications for and outcomes of emergency peripartum hysterectomy. A five-year review J Reprod Med 2000 45 733 737 11027082 Gould DA Butler-Manuel AS Turner MJ Carter PG Emergency obstetric hysterectomy-an increasing incidence J Obstet Gynaecol 1999 19 580 583 15512405 10.1080/01443619963761 Wenham J Matijevic R Post-partum hysterectomies: revisited J Perinat Med 2001 29 260 265 11447932 Nasrat HA Youssef MH Marzoogi A Talab F 'Near miss' obstetric morbidity in an inner city hospital in Saudi Arabia East Mediterr Health J 1999 5 717 726 11338695 Noor S Majid S Ruby N An audit of obstetrical hysterectomy J Coll Physicians Surg Pak 2001 11 642 645 Yamamoto H Sagae S Nishikawa S Kudo R Emergency postpartum hysterectomy in obstetric practice J Obstet Gynaecol Res 2000 26 341 345 11147720 Pattinson RC Hall MH Near misses:a useful adjunct to maternal death enquiries Br Med Bull 2003 67 231 243 14711767 10.1093/bmb/ldg007 Why mothers die 1997–1999 Report from the confidential enquiries into maternal deaths in the United Kingdom 2000 London, National Statistics Office Vandecruys HI Pattinson RC Macdonald AP Mantel GD Severe acute maternal morbidity and mortality in the Pretoria Academic Complex: changing patterns over 4 years Eur J Obstet Gynecol Reprod Biol 2002 102 6 10 12039082 10.1016/S0301-2115(01)00558-9

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==== Front BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-331534167210.1186/1471-2121-5-33Research ArticleSystemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development Yang Maocheng [email protected] Chiang Shiong [email protected] Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 1175432004 1 9 2004 5 33 33 28 6 2004 1 9 2004 Copyright © 2004 Yang and Loh; licensee BioMed Central Ltd.2004Yang and Loh; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development. Results The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C (C, DNA content of the unreplicated haploid chromosome complement) peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C. Conclusions Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed. ==== Body Background In the classical cell cycle, the nuclear DNA contents vary only within the range of 2C and 4C, where C is the haploid DNA content per nucleus. When mitotic DNA replication in somatic cells is not followed by cell division (a process called endoreduplication), variation of cellular ploidy levels (designated as somatic polyploidy or endopolyploidy) can result [1]. Endopolyploidy is considered to be developmentally regulated [2] and has been described in several plant species including maize [3,4], sunflower [5], tomato [6], Arabidopsis [7] and brassicas [8,9]. Presence of endopolyploidy as a result of endoreduplication is also a common feature of insects and mammals [10,11]. In orchids, endoreduplication has been described in the raphid crystal idioblasts of Vanilla [12] and in parenchyma cells of Vanda seedlings [13,14]. The family Orchidaceae has an estimated 17,000 to 35,000 species, making it the largest and an important family of the flowering plants [15]. Many of the members of the orchid family are commercially valuable, and are micropropagated [16]. The explant sources used for orchid micropropagation include inflorescence, leaves, floral buds and roots [16,17]. However, very little is known about the ploidy variation in different explant tissues of the orchid plants during different developmental periods and at the stage when they are used as explants for micropropagation. Increased knowledge of the degree of endopolyploidy in the explant tissue source will be highly valuable for the maintenance of the original ploidy level in culture [8]. In this paper, systemic variation of cellular ploidy and DNA content in different tissues of Spathoglottis plicata, a common tropical terrestrial orchid species, from field-grown plants and in vitro cultures was investigated. Results Ploidy level of nuclei isolated from leaves and roots of greenhouse-grown plants Flow cytometry analysis of nuclear preparations from entire young leaves of 1–3 cm in length revealed that there were three peaks of fluorescence corresponding to 2C, 4C and 8C DNA content of somatic cells (Fig. 1A). About 50% of the nuclei were found to have 2C DNA content, 25% were 4C and 15% were found to have 8C DNA content (Fig. 1G). Figure 1 Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the greenhouse-grown plants: A. Young leaves, B. Basal part of the mature leaves, C. Middle part of the mature leaves, D. Tip of the mature leaves, E. Root tips, F. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). G. The population of endopolyploid nuclei in tissues A-F. More detailed analysis was done on mature leaves of 48 cm in length. Tissues taken from different regions of the mature leaves showed that the pattern of ploidy levels was different at different regions. For tissues taken from the basal (petiolar end) of the leaves, there were three fluorescence peaks corresponding to 2C, 4C and 8C nuclear DNA content (Fig. 1B). However, no 8C peak was observed from nuclei preparations taken from tissues of the middle (Fig. 1C) and tip (Fig. 1D) regions of the same leaf; only 2C (35–40% of cell populations) and 4C (60–65% of cell population) nuclei were identified (Figs. 1C,1D,1G). In the young root tips, 2C, 4C and 8C peaks were observed (Fig. 1E) and the distribution skewed toward 2C population, which accounted for more than 60% of the nuclei population analyzed (Fig. 1G). The percentage of nuclei population with 4C and 8C DNA content in the root tip was relatively small and accounted for only about 10% each (Figs. 1E,1G). Cells from root segments taken at least 2 cm away from the tips were all 2C (Fig. 1F). Ploidy level of nuclei isolated from floral tissues Preparations from floral pedicels (Fig. 2A), columns (Fig. 2B) of freshly open flowers, and growing flower stems (Fig. 2F) revealed that there were 2C, 4C and 8C nuclei. The proportions of 2C and 4C nuclei ranged from 40–50% (Fig. 2H), and only about 8 – 10% of nuclei were found to be 8C (Fig. 2H). In pedicels of un-open flower (Fig. 2G), there was a 16C peak in addition to 2C, 4C and 8C peaks. The majority of nuclei were in 4C (28%) and 8C (46%) peaks. The 2C and 16C peaks each had less than 10% of the total nuclei (Fig. 2H). Nuclei isolated from the sepals (Fig. 2C), petals (Fig. 2D) and ovary tissues (Fig. 2E) were all 2C. Figure 2 Nuclear DNA content and distribution of endopolyploid nuclei in floral tissues of the greenhouse-grown plants: A. Pedicels, B. Columns, C. Sepals, D. Petals, E. Ovary tissues, F. Growing flower stems, G. pedicels of un-open flowers. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). H. The population of endopolyploid nuclei in tissues A-G. Ploidy level of cells from in vitro cultures Protocorms of S. plicata were found to have 2C, 4C and 8C nuclei (Fig. 3A) with majority (over 70%) of them with 2C DNA content (Fig. 3E). In the young leaves of plantlets, majority (70%) of the nuclei isolated were 4C, and about 20% of were 2C nuclei and the rest 8C (Figs. 3B,3E). In the root tips of cultures, there were about 40% each of 2C and 8C nuclei, and the proportion of 4C nuclei was only about 10% (Figs. 3C,3E). Nuclei taken from root tissues at the region with root hairs were all 2C (Figs. 3D,3E). Figure 3 Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the tissue-cultured plants: A. Protocorms, B. Young leaves, C. Root tips, D. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). E. The population of endopolyploid nuclei in tissues A-D. Discussion As references to the DNA content of gametic nucleus of individuals, DNA 'C' values have been estimated in several thousand animal and plant species [18]. For angiosperms, information on 'C' values is used in a wide range of biological fields [19]. The 1C DNA values in angiosperm plants differ approximately 1000 folds, ranging from 0.13 pg in Arabidopsis thaliana to 127.4 pg in Fritillaria assyriaca [18]. The DNA content per genome is usually considered to be constant between cells in an individual, and relatively constant between individuals of the same species [18]. However, in some plant species, intraplant ploidy variations were reported, and this implied that the nuclear DNA content in these species is not static and hence a great amount of variation occurs [8]. For example, a survey of Arabidopsis thaliana revealed endopolyploidy in hair trichomes, leaf epidermal cells, root tip cells, and cells in the hypocotyls [7,20], but not in the inflorescence [7]. In some cell types, the extent of endoreduplication appears to be intrinsically controlled by the differentiation programme, but environmental influences such as light can also affect endoreduplication [21]. The patterns of endopolyploidy may be affected by plant growth conditions in some plants. For example, leaves of in vitro grown tomato and potato plants were found to have lower level of endopolyploidy than leaves of plants grown in the greenhouse [22,23]. However, in S. plicata, patterns of endopolyploidy were found to be similar in both tissue-cultured plants and greenhouse-grown plants. Endoreduplication was found to occur in actively growing tissues with of S. plicata such as young leaves (1–3 cm in length, newly initiated) and root tips from greenhouse-grown plants. Similarly, endopolyploid cells were found in protocorms, young leaves and root tips from S. plicata seedlings in tissue culture. The common feature for protocorms, young leaves of 1–3 cm and root tips are that they are young and active in cell division and growth. In other orchids such as Dendrobium, endopolyploidy was found in root tips and newly expanded young leaves [24]. In the root segment with root hairs of S. plicata, endopolyploidy was neither found in tissue-cultured plants nor it was detected in greenhouse-grown plants. These results imply that the presence of the endopolyploidy during S. plicata development is an intrinsic programme, and it is not much affected by the growth condition. Besides root tips and newly developing young leaves, endopolyploidy was observed in mature leaves in a few Dendrobium species and cultivars [24]. Endopolyploidy was also detected in mature leaves of S. plicata. Furthermore, when the tip, middle and basal parts of the mature leaf were examined, different patterns of ploidy levels were obtained. Endopolyploidy was found only in leaf base part of mature leaves. A mature leaf represents a continuous developmental system, with the young, less green meristem cells at the basal petiolar end and the older, photosynthetically active cells at the tip [25]. Previous research shown that in cucumber and succulent plants with small genome, the level of endoreduplication does not increase once an organ is fully developed [26,27]. The tips and middle parts of mature leaves in S. plicata are fully developed. Endoreduplication in these tissues is unlikely since it would lead to further cell expansion. The pattern of DNA ploidy variation within the mature leaf is closely associated with the developmental status. The mechanism that resulted in endopolyploidy, however, remains unclear. In S. plicata, endopolyploidy was present in some floral tissues such as columns, growing flower stems and pedicels of both un-open and freshly open flowers. However, other floral tissues like sepals, petals and ovary tissues were found to have only 2C nuclei. In the growing un-open flower pedicels, the highest ploidy level even reached 16C. In cabbage, endopolyploidy was reported in cabbage flowers [8], and detailed patterns of endopolyploidy were found in various developmental stages of petals [9]. In cabbage petals, differentiation of expanding cells was characterized by endoreduplication [9]. In the proximal part of the cabbage petal, differentiation was accompanied with endoreduplication and cell enlargement. By contrast, no endopolyploid nucleus was found in the distal part of the lamina in the mature cabbage petal [9]. This study suggested that the developmental program of the cabbage petals might induce the initiation of endoreduplication [9]. In Arabidopsis, endopolyploidy was found in hypocotyls, cotyledonary leaves, rosette leaves, stems of bolting plants and floral leaves, but was not found in inflorescences [7]. Given the small size of columns within Arabidopsis floral buds, and the small population of endopolyploid nuclei found in columns and pedicels of S. plicata in this study, the minute population of the endopolyploid nuclei could easily be neglected when the whole floral buds were used for sampling. In S. plicata, it was found that the average size of nuclei was larger in columns and pedicels that have a measurable amount of endopolyploid cells than in other flora tissues without endopolyploidy (unpublished results). Similarly, a correlation was found between cell size and ploidy levels during cabbage petal development [9]. In Dendrobium, the post-pollination physiological changes were found to be different between floral tissues such as columns, ovary tissues, sepals and petals [28]. Edgar and Orr-Weaver [10] suggested that as endoreduplication is often found in large cells or cells with high metabolic activity, it might be a common strategy for cell growth without division. Further evidence was found in legumes where cell differentiation to a specialized function as pod wall tissues was accompanied by endoreduplication, and higher ploidy levels coincided with maximum pod growth [29]. During tomato fruit development, the pericarp tissue of young green fruit did not have higher ploidy (usually within 2C and 4C), but most of the cells in pericarp became endopolyploid (up to 256C) as the fruit developed further [6]. In tobacco single cell culture, endoreduplication was associated with plant growth regulators. When auxin was applied alone, endoreduplication was induced and the DNA content kept pace with the increment of cell volume. When both auxin and cytokinin were supplied subsequently, the cells divided first as amitosis leading to DNA endoreduplication, then followed by normal mitosis cell cycles [30]. Gibberellin and ethylene were found to play important roles in the endoreduplication of Arabidopsis hypocotyls [31]. In cabbage, mammals, Drosophila melanogaster and some small genome plants like Arabidopsis, it is thought that endoreduplication is developmentally regulated [8,10,32]. The systemic endopolyploidy revealed within different tissues of S. plicata raises the question of its possible implications. In tobacco, it was reported that the morphogenetic response of the tissues culture was related to the nuclear DNA content variation within stem explants of different ages [25]. In Oncidium Gower Ramsey, a hybrid orchid, only root tips, cut surfaces of stem segments and young leaves were able to form callus in tissue culture. Other explants such as old leaves and the roots without meristem tips could not form any callus [33]. Molecular data showed that the nuclear DNA modulation was closely related to the acquisition of embryogenic competence in cultured carrot hypocotyls [34]. In various tissues in cabbage plants, the number of endocycles was tissue-specific and was characteristic of the developmental stage [8,9,32]. These studies suggested that pattern of endopolyploidy may represent the characteristic of the developmental and physiological properties of the tissue. The role of endoreduplication in plant development is still not well understood. The presence of endopolyploidy was proposed to be associated with several factors, such as taxonomic position of a species, life cycle, genome size, and organ type [35]. Recent investigation of 16 plant species suggested that endopolyploidization might provide a mechanism to facilitate plant growth [35]. Endoreduplication benefits fast growth in several ways. In polyploid cells, the increased gene dosages may enhance the transcriptional and metabolic activities. In addition, several processes are eliminated in the endoreduplication cycle such as the reorganization of the cytoskeleton and condensation of the chromosomes, and that might allow faster growth [36]. Ploidy level also plays a role in controlling the size of the cells, the organs or the whole plant [6,9,37]. One of the common features of plant development is the uneven enlargement of plant cells coupled to somatic endoreduplication, which indicates that the enlargement of plant cells might be the consequence of the increased genome size [9,37]. This research may also have an impact on the orchid industry. Orchidaceae is the largest family of the flowering plants, and many of its members are commercially hybridized [38]. Clonal propagation is a common and essential practice for multiplication of hybrid orchids because the genotypes of the hybrids are usually heterozygous [39]. Many tissues have been used as explants for micropropagation including inflorescence, leaves, floral buds and roots [16,40,41]. Somaclonal variation is undesirable, and it is a major problem encountered in commercial micropropagation of orchids if true-to-type plants are required [39,42]. The mechanism of the somaclonal variation is poorly understood [42]. Polyploidy is considered as a possible cause for somaclonal variation in tissue cultures [43], but how polyploidy is generated during tissue culture is unclear [39,42]. The presence of systemic endopolyploidy and DNA content variation within different tissues of S. plicata as revealed in this study suggests that endopolyploidy and DNA content variation in explants might be a cause for somaclonal variation in tissue culture derived orchid plantlets. Thus, the pre-knowledge about the ploidy variation in different explant tissues is valuable for clonal propagation or for deliberate induction of variants in culture. Further systemic investigation of the relationship between somaclonal variation and type and endopolyploid level of source explants will provide indepth knowledge for micropropagation of orchids. Conclusions Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was developmentally regulated. Pattern of endopolyploidy is a character of the developmental and physiological properties of the tissue. This finding provides useful information for understanding of the plant development and for industrial propagation of orchids. Methods Plant materials Spathoglottis plicata L. is a common tropical terrestrial orchid. The plants were grown in pots and placed in the greenhouse at 28 ± 4°C without artificial lighting. The following materials were taken for analysis: a) young leaves (1–3 cm in length), b) mature leaf (48 cm in length), c) root tips (2 cm including the tip), d) root segments from region with root hairs (2 cm away from the root tip), e) newly opened flowers, f) growing flower stems and g) developing floral pedicels of un-open flower (4–6 days before flowering). Seedpods were surface-sterilized for 20 min in 20% (v/v) Clorox™ solution and subsequently rinsed 3 times with autoclaved water. Seeds were germinated aseptically in 9 cm diameter petri dishes containing 25 ml Knudson C orchid medium (Duchefa, Netherlands) with 2% (w/v) sucrose and 0.8% (w/v) agar. All cultures were incubated at 25 ± 2°C under a 16 h photoperiod (light intensity: 54 μm-1m-2 s-1). The following materials from in vitro cultures were examined: a) protocorms (6 weeks after germination), b) young leaves (1–2 cm in length) from seedlings, c) root tips (1 cm segments from tips), d) root segments with root hairs (at least 1 cm away from the root tips). Preparation of nuclei and flow cytometry analysis of nuclear DNA content Extraction of nuclei and staining of DNA were performed according to the method of Arumuganathan and Earle [44,45] with some modifications. All preparations were done on ice. Tissues (about 0.3 – 1.0 g) were sliced with razor blades into strips of less than 1 mm in 1 ml extraction solution (1 mM MgSO4, 5 mM KCl, 0.5 mM HEPES, 1 mg/ml dithiothreitol, 2.5 mg/ml Triton X-100, pH 8.0) and extracted for 45 minutes. After filtering through a 45 μm Falcon cell strainer, 100 μl of propidium iodide (1 mg/ml) and 2.5 μl of 500 μg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN) were added to each sample followed by 30 min incubation at 37°C. A Coulter EPICS® Elite ESP with 15 mW 488 nm Cyonics Argon air-cooled laser flow cytometer was used to measure the relative fluorescence of nuclei. For each sample, at least 10,000 nuclei were analyzed. Data were analyzed with WinMDI27B software (Joseph Trotter™). Abbreviations C, DNA content of the unreplicated haploid chromosome complement PI, propidium iodide PMT4, photo multiplier tube Authors' contributions MY carried out experiments in the project. CSL and MY prepared the manuscript. Acknowledgement The authors thank M. S. Choy for his help on flow cytometry. ==== Refs Larkins BA Dikes BP Dante RA Coelho CM Woo Y-M Liu Y Investigating the hows and whys of DNA endoreduplication J Exp Bot 2001 52 183 192 11283162 10.1093/jexbot/52.355.183 Joubes J Chevalier C Endoduplication in higher plants Plant Mol Biol 2000 43 735 745 11089873 10.1023/A:1006446417196 Schweizer L Yerk-Davis GL Phillips RL Srienc F Jones RJ Dynamics of maize endosperm development and DNA endoreduplication Proc Natl Acad Sci USA 1995 92 7070 7074 7624371 Setter TL Flannigan BA Water deficit inhibits cell division and expression of transcripts involved in cell proliferation and endoreduplication in maize endosperm J Exp Bot 2001 52 1401 1408 11457899 10.1093/jexbot/52.360.1401 Price HJ Johnston JS Influence of light on DNA content of Helianthus annuus Linnaeus Proc Natl Acad Sci USA 1996 93 11264 11267 8855344 10.1073/pnas.93.20.11264 Bergervoet JHW Verhoeven HA Gilissen LJW Bino RJ High amount of nuclear DNA in tomato (Lycopersicon esculentum Mill.) pericarp Plant Sci 1996 116 141 145 10.1016/0168-9452(96)04383-X Galbraith DW Harkins KR Knapp S Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiol 1991 96 985 989 16668285 Kudo N Kimura Y Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species Theor Appl Genet 2001 102 104 110 10.1007/s001220051624 Kudo N Kimura Y Nuclear DNA endoreduplication during petal development in cabbage: relationship between ploidy levels and cell size J Exp Bot 2002 53 1017 1023 11971913 10.1093/jexbot/53.371.1017 Edgar BA Orr-Weaver TL Endoreduplication cell cycles: more or less Cell 2001 105 297 306 11348589 10.1016/S0092-8674(01)00334-8 Nagl W DNA endoreduplication and polyteny understood as evolutionary strategies Nature 1976 261 614 615 934306 Kausch AP Horner HT Increased nuclear DNA content in raphide crystal idioblasts during development in Vanilla planifolia L. (Orchidaceae) Eur J Cell Biol 1984 33 7 12 6698045 Alvarez MR Sagawa Y A histochemical study of embryo development in Vanda (Orchidaceae) Caryologia 1965 18 251 261 Alvarez MR Quantitative changes in nuclear DNA accompanying postgermination embryonic development in Vanda (Orchidaceae) Am J Bot 1968 55 1036 1041 Dressler RL Phylogeny and classification of the orchid family 1993 Portland, Oregon, Dioscorides Press 314 318 Arditti J Arditti J Clonal propagation of orchids by means of tissue culture-a manual Orchid Biology, Reviews and Perspectives I 1977 Ithaca and London, Comstock Publishing Associates 203 295 Intuwong O Sagawa Y Clonal propagation of Sarcanthine orchids by aceptic culture of influorescences Am Orchid Soc Bull 1973 42 209 215 Hanson L McMahon K Johnson MT Bennett MD First nuclear DNA C-values for 25 angiosperm families Ann Bot (Lond) 2001 87 251 258 10.1006/anbo.2000.1325 Bennett BD Leitch IJ Nuclear DNA amounts in angiosperms Ann Bot (Lond) 1995 76 113 176 10.1006/anbo.1995.1085 Melaragno JE Mehrotra B Coleman AW Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis Plant Cell 1993 5 1661 1668 12271050 10.1105/tpc.5.11.1661 Traas J Holskamp M Gendreau E Hofte H Endoreduplication and develoment: rule without dividing? Curr Opin Plant Biol 1998 1 498 503 10066638 10.1016/S1369-5266(98)80042-3 Smulders MJM Rus-Kortekaas W Gilissen LJW Development of polysomaty during differentiation in diploid and tetraploid tomato (Lycopersicon esculentum) plants Plant Sci 1994 97 53 60 10.1016/0168-9452(94)90107-4 Uijtewaal BA Ploidy variability in greenhouse cultured and in vitro propagated potato (Solanum tuberosum) monohaploids (2n = x = 12) as determinated by flow cytometry Plant Cell Rep 1987 6 252 255 Jones WE Kuehnle AR Ploidy identification using flow cytometry in tissues of Dendrobium species and cultivars Lindleyana 1998 13 11 18 10.1017/S0268416098003051 Altamura MM Bassi P Cavallini A Gionini G Cremonini R Monacelli B Pasqua G Sassoli O Tran Thanh Van K Cionini PG Nuclear DNA changes during plant development and the morphogenetic response in vitro of Nicotiana tobacum tissues Plant Sci 1987 53 73 79 10.1016/0168-9452(87)90180-4 De Rocher EJ Harkins KR Galbraith DW Bohnert HJ Developmentaly regulated systemic endopolyploidy in succulents with small genomes Science 1990 250 99 101 Gilissen LJW van Staveren MJ Creemers-Molenaar J Verhoeven HA Development of polysomaty in seedlings and plants of Cucumis sativus L Plant Sci 1993 91 171 179 10.1016/0168-9452(93)90140-U Ketsa S Rugkong A Senescence of Dendrobium 'Pompadour' flowers following pollination J Hortic Sci Biotechnol 1999 74 608 613 Lagunes-Espinoza LC Huyghe C Bousseau D Barre P Papineau J Endoreduplication occurs during pod wall development in temperate grain legumes Ann Bot (Lond) 2000 86 185 190 10.1006/anbo.2000.1174 Valente P Tao W Verbelen JP Auxins and cytokinins control DNA endoreduplication and deduplication in single cells of tobacco Plant Sci 1998 134 207 215 10.1016/S0168-9452(98)00062-4 Gendreau E Orbovic V Höfte H Traas J Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyls Planta 1999 209 513 516 10550633 10.1007/s004250050755 Kudo N Kimura Y Flow cytometric evidence for endopolyploidization in cabbage (Brassica oleracea L.) flowers Sex Plant Reprod 2001 13 279 283 10.1007/s004970100066 Chen JT Chang WC Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae) Plant Sci 2000 160 87 93 11164580 10.1016/S0168-9452(00)00367-8 Geri C Turrini A Giorgetti L Nicoletti E Ronchi VE Genome plasticity during the acquisition of embryogenic competence Genome 1999 42 1134 1143 10659781 10.1139/gen-42-6-1134 Barow M Meister A Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size Plant Cell Environ 2003 26 571 584 Kondorosi E Kondorosi A Endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development FEBS Letters 2004 567 152 157 15165909 Kondorosi E Roudier F Gendreau E Plant cell-size control: growing by ploidy? Curr Opin Plant Biol 2000 3 488 492 11074380 10.1016/S1369-5266(00)00118-7 Hew CS Young JWH Hew CS, Young JWH The relevance of orchid physiology to the industry The physiology of tropical orchids in relation to the industry 1997 Singapore, World Scientific 1 11 Vajrabhaya T Arditti J Variations in clonal propagation Orchid Biology, Reviews and Perspectives I 1977 Ithaca and London, Comstock Publishing Associates 179 201 Arditti J Krikorian AD Orchid micropropagation: the path from laboratory to commercialization and an account of several unappreciated investigations Bot J Linn Soc 1996 122 183 241 10.1006/bojl.1996.0059 Teng WL Nicholson L Teng MC Micropropagation of Spathoglottis plicata Plant Cell Rep 1997 16 831 835 10.1007/s002990050329 Ichihashi S Arditti J, Pridgeon AM Variations in clonal propagation Orchid Biology, reviews and perspectives, VII 1997 Dordrecht, Boston and London, Kluwer Academic Publishers 285 306 D'Amato F Cytogenetics of plant cell and tissue cultures and their regenerates CRC crit rev plant sci 1985 3 73 112 Arumuganathan K Earle ED Estimation of nuclear DNA content of plants by flow cytometry Plant Mol Biol Rep 1991 9 229 241 Arumuganathan K Earle ED Nuclear DNA content of some important plant species Plant Mol Biol Rep 1991 9 208 218

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==== Front BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-5-261533934310.1186/1471-2156-5-26Research ArticleMost of the extant mtDNA boundaries in South and Southwest Asia were likely shaped during the initial settlement of Eurasia by anatomically modern humans Metspalu Mait [email protected] Toomas [email protected] Ene [email protected] Jüri [email protected] Georgi [email protected] Katrin [email protected] Piia [email protected] Monika [email protected] Doron M [email protected] M Thomas P [email protected] Phillip [email protected] Sarabjit [email protected] Surinder S [email protected] Karl [email protected] Antonio [email protected] Richard [email protected] Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia2 Bruce Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa, Israel3 Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy4 Department of Human Sciences, Loughborough University, Loughborough, United Kingdom5 Department of Human Genetics, University of Newcastle-upon-Tyne, United Kingdom6 Ecology and Evolutionary Biology, The University of Arizona, Tucson, Arizona, USA7 Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, Oxford OX1 3PS,United Kingdom2004 31 8 2004 5 26 26 7 5 2004 31 8 2004 Copyright © 2004 Metspalu et al; licensee BioMed Central Ltd.2004Metspalu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian (including both tribal and caste populations from different parts of India) and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia. Results Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades. Conclusions Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent. ==== Body Background Two mtDNA macro-haplogroups (M and N) that arose from the African haplogroup L3 encompass virtually all mtDNAs outside Africa [1-4]. The phylogenetic node N (including R) has spread its branches all over Eurasia, in contrast to haplogroup M, which is found in Eastern Eurasia but is virtually absent in Europe. The numerous branches of N are, however, generally segregated to either the eastern (e.g. A, B [5], Y [6], R9 [7] or western (e.g. N1 [8,9], N2 (comprising of W and its sister-clade identified by [10]), TJ, HV, U [11]) Eurasian-specific pools. The majority of Indian mtDNAs belong to macro-haplogroup M [8,12-21]. While the topology of the M sub-haplogroups that are common in mainland East Asia (M7, M8 (including C, Z), M9 (including E), D, G [7,22,23]) and in Africa (M1 [24]) is established in detail, the internal haplogroup structure of M in India has remained largely undefined. We have previously demonstrated that transitions at nps. 477G, 1780, 8502 and 16319 designate Indian-specific haplogroup M2, the most frequent M clade in India [15]. Another Indian-specific M clade supported by HVS-I variation as well as coding region markers, is M6 [15]. Haplogroups M3, M4 and M5 have been discriminated preliminarily by their characteristic HVS-I mutations [19], but since their defining positions, 16126, 16311, and 16129, respectively, are phylogenetically unstable [25,26], it is unlikely that the proposed haplogroups are monophyletic. Most numerous sub-groups of macro-haplogroup N in India are the Indian-specific variants of the phylogenetic node R including haplogroups R5, R6, U2(a, b, c) [8,13,27]. The overwhelming majority of the Iranian mtDNAs have been shown to lie in the West Eurasian domain of the global human mtDNA pool [27,28]. Here we focus on the analysis of mtDNA lineages that are shared between Indians and Iranians and bear signals of pre-Holocene expansion in the region. India congregates four linguistic domains (Indo-European, Dravidic, Austro-Asiatic and Tibeto-Burman) that occupy non-random spheres of the geographic distribution of its populations. The majority of the recent studies based on mtDNA variation have, in contrast to some [21], provided evidence that linguistic groups of India do not represent genetically homogeneous units and are not, therefore, traceable to different immigration waves from distinct sources [8,13,19]. The complexity that arises in defining populations and groups of populations in India based on genetic and cultural criteria has been recently demonstrated in South Indian tribal and caste populations. The combined data from mtDNA, Y-chromosome and autosomal genes indicated that the tribes and castes derive largely from the same genetic heritage of Late Pleistocene southern and southwestern Asians, and have received limited gene flow from external sources since the Holocene [15]. Similar results were obtained by Cordaux et al. [29], who demonstrated that caste and tribal groups exhibit similar levels of molecular variance. However, genetic distances indicated that the Tibeto-Burman speaking tribal populations (from eastern India) were more closely related to East Asians than to other Indians [29]. This is consistent with an earlier suggestion placing the origin of these tribal groups east of India – in Tibet and Myanmar [30]. In this study, we have analyzed the mtDNA variation in a sample of 796 Indians and 436 Iranians (Table 1), and combined the results with previously published data from the same geographic area. We also compared the mtDNA variation in India and Iran with that of Europe, China, and Thailand. The overall aim was to improve our understanding of the origins and composition of the Indian and Iranian gene pools and to determine the nature and the extent of gene flow between these regions. Through the analyses of the genetic variation of extant Southwest and South Asian populations we took an endeavor to envisage the exodus of anatomically modern humans from Africa. Table 1 Characteristics of the Indian and Iranian population samples whose mtDNA variation has been determined in the course of this study. INDIA State / Region Socio-cultural affiliation Linguistic affiliation Population Code n N West Bengal Caste Indo-European Mixed caste people Ben 50 80 Million Uttar Pradesh Tribe Indo-European Bhoksa Bho 5 32,000 Kerala Caste Indo-European Mixed caste people from Cochin Co 55 600,000 Kerala -a Indo-European Cochin Jews CoJ 45 5,000 (in Israel) Gujarat Caste Indo-European Mixed caste people Guj 53 50 Million Himachal Tribe Tibeto-Burman Kanet Kan 37 33,000 Maharashtra Caste Indo-European Konkanastha Brahmin Kon 58 N/A West Bengal Caste Indo-European Kurmi Kur 55 N/A West Bengal Tribe Austro-Asiatic Lodha Lod 56 59,000 Sri Lanka Caste Dravidic Moor Mo 50 3.4 Million Maharashtra -a Indo-European Parsi Par 55 76,000 Punjab Caste Indo-European Mixed caste people Pun 109 24 Million Rajasthan Caste Indo-European Rajput Raj 35 5 Million (in Rajasthan) Sri Lanka Caste Indo-European Sinhalese Sin 82 14.6 Million Uttar Pradesh Tribe Indo-European Tharu Tha 26 96,000 (in India) Uttar Pradesh Caste Indo-European Uttar Pradesh Brahmin UPb 25 2.5 Million total 796 IRAN northwest 226 southwest 138 northeast 30 southeast 6 central 36 total Indo-European Iranian IR 436 ca. 68 Million a For some analyzes these populations where grouped with the caste populations. (see Table 1 in the Supplementary Material for the complete set of studied populations including those whose mtDNA variation was previously published) Results and Discussion Geographic distribution of macro-clades M and N in India We found haplogroup M ubiquitous at almost 58% among the caste, and 72% among the tribal populations (Table 2), which is largely consistent with previous reports [8,12-21]. Our results indicate that the frequency distribution of haplogroup M varies across different Indian regions by a significant cline towards the south and the east (see Figure 4 for Spatial Autocorrelation Analysis (SAA) p < 0.05). The variation among caste populations climbs from approximately 40% in Gujarat and (Indian-) Punjab to 65% in the southern states, and peaks at over 70% in West Bengal (Table 2). We observed a similar geographic pattern among tribal populations, where the frequency varied from just over 50% in the northern states of Punjab and Himachal Pradesh, increased to 70%–80% in the southern states, and peaked at 86% in West Bengal (Table 8, see Additional file 3). Figure 4 Spatial Autocorrelation Analyses Correlograms of different haplogroups and haplogroup groupings frequencies in South Asia. In the case of haplogroup M3a some datasets had to be excluded because lack of resolution (see footenote for Table 3. for detailes). There was no significant cline in the frequencies of haplogroup R2. In the case of haplogroups M6a and M6b only the potential cline along the Bay of Bengal is investigated. Therefor the number of distance classes is reduced to three. Table 2 Geographic, linguistic and socio-cultural distribution of major Indian-specific mtDNA haplogroups HAPLOGROUP FREQUENCY (95% CR FOR PROPORTION) n Mb M2 M3a M4a M6 M18 M25 Rc R5 R6 U2id R2; U7; W EEe WEf India 2544g 65 (63.1–66.8) 6.3 (5.4–7.3) 2.2 (1.7–2.9) 0.6 (.4–1) 2.9 (2.3–3.6) 2.1 (1.6–2.8) 0.9 (.6–1.4) 11.4 (10.2–12.7) 2.2 (1.7–2.8) 1.3 (.9–1.8) 5.3 (4.5–6.2) 3.4 (2.7–4.2) 10.5 (9.4–11.8) 12.1 (10.9–13.4) Iran 436 5.3 (3.6–7.8) 0 (0–.8) 0.5 (.1–1.6) 0 (0–.8) 0 (0–.8) 0.2 (.1–1.3) 0 (0–.8) 3.2 (1.9–5.3) 0.9 (.4–2.3) 0 (0–.8) 0.2 (.1–1.3) 13.3 (10.4–16.8) 3.9 (2.5–6.2) 90.8 (87.7–93.2) Socio-cultural affiliation (Indian data only) Caste population 1204 57.8 (54.9–60.5) 5.1 (4–6.5) 4.2 (3.2–5.5) 1.1 (.6–1.8) 3 (2.2–4.1) 1.4 (.9–2.2) 1.4 (.9–2.2) 14.1 (12.3–16.2) 3.3 (2.5–4.5) 1.7 (1.1–2.5) 7.8 (6.4–9.5) 4.8 (3.7–6.2) 4 (3–5.2) 17.1 (15.1–19.3) Tribal population 1332 71.5 (69–73.8) 7.5 (6.2–9) 0.5 (.2–1) 0.2 (0–.5) 2.7 (2–3.7) 2.7 (2–3.7) 0.5 (.2–1) 8.9 (7.5–10.6) 1.2 (.7–1.9) 0.9 (.5–1.6) 3 (2.2–4.1) 2 (1.4–2.9) 16.5 (14.6–18.6) 7.5 (6.2–9) Brahmins & Kshatriyas 313 60.1 (54.5–65.3) 1.9 (.9–4.1) 5.1 (3.2–8.1) 1 (.3–2.8) 0.6 (.2–2.3) 1 (.3–2.8) 2.2 (1.1–4.5) 14.7 (11.2–19.1) 4.5 (2.7–7.4) 1.3 (.5–3.2) 6.4 (4.2–9.7) 6.1 (3.9–9.3) 3.5 (2–6.2) 17.6 (13.8–22.2) Other Castesh 517 61.5 (57.2–65.6) 6.2 (4.4–8.6) 4.6 (3.1–6.8) 1.2 (.5–2.5) 2.9 (1.8–4.7) 2.1 (1.2–3.8) 0.8 (.3–2) 12.6 (10–15.7) 2.3 (1.3–4) 1.5 (.8–3) 7.4 (5.4–9.9) 3.9 (2.5–5.9) 1.7 (.1–1.4) 16.6 (1.2–3.8) Language groups of India Indo-European 963 57.5 (54.4–60.6) 3.8 (2.8–5.3) 4.5 (3.3–6) 1.0 (.6–1.9) 2.7 (1.9–3.9) 1.1 (.6–2) 1.6 (1–2.6) 7.8 (6.3–9.7) 2.5 (1.7–3.7) 1.6 (1–2.6) 7.1 (5.6–8.9) 6.9 (5.4–8.6) 4.0 (3–5.5) 19.9 (17.5–22.6) Dravidic 1063 70.0 (67.2–72.7) 10.8 (9.1–12.8) 1.2 (.7–2.1) 0.5 (.2–1.1) 4.0 (2.9–5.3) 1.4 (.9–2.3) 0.5 (.2–1.1) 8.9 (7.4–10.8) 2.7 (1.9–3.9) 1.4 (.9–2.3) 5.9 (4.7–7.5) 1.2 (.7–2.1) 3.0 (2.1–4.2) 8.8 (7.3–10.7) Tibeto-Burman 249 59.8 (53.6–65.7) 0 (0–1.5) 0.4 (.1–2.2) 0 (0–1.5) 1.6 (.7–4) 1.2 (.4–3.5) 1.2 (.4–3.5) 0.8 (.2–2.9) 1.2 (.4–3.5) 0 (0–1.5) 0.8 (.2–2.9) 2.0 (.9–4.6) 61.4 (55.3–67.3) 3.2 (1.7–6.2) Austro-Asiatic (Mundari) 90 86.7 (78.1–92.2) 2.2 (.7–7.7) 0 (0–4) 0 (0–4) 0 (0–4) 26.7 (18.6–36.7) 0 (0–4) 4.4 (1.8–10.9) 0 (0–4) 1.1 (.3–6) 1.1 (.3–6) 1.1 (.3–6) 0 (0–4) 1.1 (.3–6) Indo-European i 250 66.4 (60.3–72) 8.0 (5.3–12) 0.4 (.1–2.2) 0.8 (.2–2.8) 4.8 (2.8–8.2) 1.6 (.6–4) 0.4 (.1–2.2) 8.8 (5.9–13) 2.0 (.9–4.6) 1.6 (.6–4) 6.4 (4–10.1) 4.0 (2.2–7.2) 1.2 (.4–3.5) 13.6 (9.9–18.4) Indo-European j 713 54.4 (50.7–58) 2.4 (1.5–3.8) 5.9 (4.4–7.9) 1.1 (.6–2.2) 2.0 (1.2–3.3) 1.0 (.5–2) 2.0 (1.2–3.3) 7.4 (5.7–9.6) 2.7 (1.7–4.1) 1.5 (.9–2.7) 7.3 (5.6–9.4) 7.9 (6.1–10.1) 5.0 (3.7–6.9) 22.2 (19.3–25.4) Indian States and Bangladesh (excluding tribal populations)k Andhra Pradesh 245 65.3 (59.1–71.0) 7.7 (5–11.8) 2.8 (1.4–5.7) 1.6 (.6–4.1) 3.3 (1.7–6.4) 4.1 (2.3–7.4) 0 (0–1.5) 18 (13.7–23.2) 4.1 (2.3–7.4) 1.2 (.4–3.5) 5.3 (3.2–8.9) 2.4 (1.1–5.2) 1.2 (.4–3.5) 8.9 (6–13.2) Bangladesh 30 66.67 (48.6–80.8) 9.96 (3.61–25.7) 0 (.08–11.2) 0 (.08–11.2) 3.29 (.78–16.6) 3.29 (.78–16.6) 0 (.08–11.2) 6.67 (2.04–21.4) 0 (.08–11.2) 0 (.08–11.2) 6.67 (2.04–21.4) 3.29 (.78–16.6) 9.96 (3.61–25.7) 6.67 (2.04–21.4) Gujarat 57 40.4 (28.6–53.4) 5.3 (1.9–14.4) 7 (2.9–16.7) 1.8 (.4–9.2) 1.8 (.4–9.2) 1.8 (.4–9.2) 0 (0–6.2) 17.5 (9.9–29.4) 5.3 (1.9–14.4) 1.8 (.4–9.2) 1.8 (.4–9.2) 17.5 (9.9–29.4) 5.3 (1.9–14.4) 33.3 (22.5–46.3) Karnataka 47h 55.3 (41.2–68.6) 2.1 (.5–11.1) 2.1 (.5–11.1) 0 (.1–7.4) 0 (.1–7.4) 0 (.1–7.4) 0 (.1–7.4) 19.1 (10.5–32.6) 6.4 (2.3–17.2) 0 (.1–7.4) 10.6 (4.7–22.7) 6.4 (2.3–17.2) 0 (.1–7.4) 14.9 (7.5–27.8) Kashmir 19 26.3 (11.9–49.1) 0 (.1–16.8) 0 (.1–16.8) 5.3 (1.2–24.9) 10.5 (3.2–31.7) 0 (.1–16.8) 0 (.1–16.8) 10.5 (3.2–31.7) 0 (.1–16.8) 5.3 (1.2–24.9) 10.5 (3.2–31.7) 5.3 (1.2–24.9) 21.1 (8.7–43.7) 31.6 (15.4–54.3) Kerala 100 54 (44.2–63.5) 4 (1.6–9.8) 0 (0–3.6) 0 (0–3.6) 0 (0–3.6) 0 (0–3.6) 5 (2.2–11.2) 25 (17.6–34.3) 9 (4.9–16.2) 1 (.2–5.4) 7 (3.5–13.8) 1 (.2–5.4) 4 (1.6–9.8) 11 (6.3–18.7) Maharashtra 117 65 (55.9–73) 3.4 (1.4–8.5) 11.1 (6.6–18.1) 0.9 (.2–4.6) 0 (0–3.1) 0 (0–3.1) 5.1 (2.4–10.7) 6.8 (3.5–12.9) 0.9 (.2–4.6) 0.9 (.2–4.6) 4.3 (1.9–9.6) 4.3 (1.9–9.6) 5.1 (2.4–10.7) 23.1 (16.4–31.5) Punjab 150 36 (28.8–44) 0.7 (.2–3.6) 4.7 (2.3–9.3) 1.3 (.4–4.7) 0.7 (.2–3.6) 0 (0–2.4) 2.7 (1.1–6.6) 11.3 (7.2–17.4) 0.7 (.2–3.6) 0.7 (.2–3.6) 7.3 (4.2–12.7) 14 (9.4–20.5) 5.3 (2.8–10.2) 42.7 (35–50.7) Rajasthan 36 69.4 (53–82) 0 (.1–9.5) 13.9 (6.2–28.8) 0 (.1–9.5) 0 (.1–9.5) 5.6 (1.7–18.2) 0 (.1–9.5) 22.2 (11.8–38.2) 5.6 (1.7–18.2) 2.8 (.7–14.2) 2.8 (.7–14.2) 5.6 (1.7–18.2) 0 (.1–9.5) 2.8 (.7–14.2) Sri Lanka 132 57.6 (49–65.7) 6.8 (3.7–12.5) 1.5 (.5–5.3) 1.5 (.5–5.3) 1.5 (.5–5.3) 0.8 (.2–4.1) 0 (0–2.7) 12.9 (8.2–19.7) 0.8 (.2–4.1) 3 (1.2–7.5) 12.1 (7.6–18.8) 2.3 (.8–6.5) 2.3 (.8–6.5) 15.2 (10–22.3) Tamil Nadu 51 64.7 (50.9–76.4) 5.9 (2.1–15.9) 3.9 (1.2–13.2) 2 (.5–10.3) 2 (.5–10.3) 2 (.5–10.3) 0 (0–6.8) 11.8 (5.6–23.4) 0 (0–6.8) 3.9 (1.2–13.2) 2 (.5–10.3) 3.9 (1.2–13.2) 23.5 (14–36.8) 7.8 (3.2–18.5) Uttar Pradesh 98 52 (42.2–61.7) 1 (.2–5.5) 8.2 (4.2–15.3) 1 (.2–5.5) 0 (0–3.7) 2 (.6–7.1) 1 (.2–5.5) 11.2 (6.4–19) 3.1 (1.1–8.6) 2 (.6–7.1) 15.3 (9.5–23.8) 3.1 (1.1–8.6) 4.1 (1.7–10) 13.3 (8–21.4) West Bengal 106 71.7 (62.5–79.4) 3.8 (1.5–9.3) 1.9 (.6–6.6) 0 (0–3.4) 5.7 (2.7–11.8) 0 (0–3.4) 0.9 (.2–5.1) 12.3 (7.3–19.9) 6.6 (3.3–13) 2.8 (1–8) 6.6 (3.3–13) 0.9 (.2–5.1) 0.9 (.2–5.1) 7.5 (3.9–14.2) a a See Tables 8–13 (additional files 3–7, 9) for frequencies of other haplogroups including breakdown of R2, U7 and W b Includes all haplogroup M sub-haplogroups c Includes sub-haplogroups R2, R5, R6 and R10 d Composite of haplogroups U2a, U2b and U2c [27] e Composite of haplogroups A, B, D, G, M7, M8 (including C and Z), M9 (including E), M10, N9 (including Y), R9 (including F) f Composite of haplogroups HV, TJ, X, N1, N2 and U (except U2i) g Caste and tribal populations plus 8 Siddi [49] who belong to neither groupings h Datasets where caste affiliation was not determined (undefined/mixed caste population), Scheduled Caste populations (e.g. the Mukri, see text for explanation) and data from [3] (dataset of 27 haplogroup M samples) are excluded. i Indo-European speakers of southern India (Tamil Nadu, Kerala, Karnataka, Andhra Pradesh and Sri Lanka) j Indo-European speakers of northern India (States other than listed in the previous footnote) k The sum of the presented caste populations sample sizes is different from that given in the third row of the current table because of the addition of the Bangladeshi (n = 30) and extraction of the Mukri (n = 42), Orissa (n = 2) and Bihar (n = 2) data. With the exception of the diverse set of largely Indian-specific R lineages, the most frequent mtDNA haplogroup in India that derives from the phylogenetic node N is haplogroup W [13]. The frequency peak of haplogroup W is 5% in the northwestern states – Gujarat, Punjab and Kashmir. Elsewhere in India its frequency is very low (from 0 to 0.9%) (Table 2) forming a significant spatial cline (Figure 4). At 15% among the caste and 8% among the tribal populations haplogroup U is the most frequent sub-clade of R in India (Table 12, see Additional file 7). Approximately one half of the U mtDNAs in India belong to the Indian-specific branches of haplogroup U2 (U2i: U2a, U2b and U2c) [13,27] (Table 2). They are present throughout India without a clear geographical cline (Figure 2, panel U2i, SAA p > 0.05). However, the spread of another subset of U, haplogroup U7 [13], is similar to that of haplogroup W, peaking at 12% and 9% in Gujarat and Punjab, respectively (Table 11, see Additional file 6). The frequency of U7 is also high in neighboring Pakistan (6%) and particularly in Iran (9%) (Table 9, see Additional file 4). Figure 2 The spatial distribution of Indian-specific mtDNA haplogroups (U2, M4a, M18 and M25) and their sub-haplogroups. In the case of haplogroup M25 some datasets had to be excluded because the discrimination between M* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). For other details see legend to Figure 1. MtDNA haplogroups in Iran Over 90% of the mtDNAs found in Iran belong to haplogroups HV, TJ, U, N1, N2 and X, commonly found in West Eurasia (Table 2). In contrast to Europe, where H is predominant among the mtDNA haplogroups, in Iran the frequency of haplogroup U (29%) is higher than that of haplogroup H (17%) (Table 9, see Additional file 4). This difference accounts, at least partly for the presence in Iran of U sub-groups, such as U7 (9.4%), that are virtually absent in Europe. Compared to India, haplogroup M frequency in Iran is marginally low (5.3%) and there are no distinguished Iranian-specific sub-clades of haplogroup M. All Iranian haplogroup M lineages can be seen as derived from other regional variants of the haplogroup: eleven show affiliation to haplogroup M lineages found in India, twelve in East and Central Asia (D, G, and M8) and one in northeast Africa (M1). Indian-specific (R5 and Indian-specific M and U2 variants) and East Asian-specific (A, B and East Asian-specific M subgroups) mtDNAs, both, make up less than 4% of the Iranian mtDNA pool. We used Turkey (88.8 ± 4.0%) as the third parental population for evaluating the relative proportions of admixture from India (2.2 ± 1.7%) and China (9.1 ± 4.1%) into Iran. Therefore we can conclude that historic gene flow from India to Iran has been very limited. The package of the most ancient mtDNA haplogroups in India Approximately one tenth of the Indian haplogroup M mtDNAs fall into its major sub-clade M2, which is defined by the motif 477G-1780-8502-16319 [15]. M2 can be further subdivided into haplogroups M2a (transitions at nps 5252 and 8369) and M2b [15]. Haplogroup M2 and its two major sub-clades reveal coalescence times of 50 to 70 thousand years (Table 3). Due to the increased frequency towards the southern part of India (Figure 1, panel M2, SAA p < 0.05 Figure 4), M2 is significantly (p < 0.05) more frequent among the Dravidic speakers than among the Indo-European speakers who are spread mostly in the northern regions of India (Table 2). It is more plausible that geography rather than linguistics is behind this pattern, because the frequency of M2 amongst the Indo-European speaking populations in southern India is significantly higher than that in the north, while there is no significant difference between Dravidic and Indo-European speaking populations from the same geographic region (Table 2). It is also notable that the frequency of M2 among the Brahmins and the Kshatriyas of Andhra Pradesh (CR 3.3 – 19.2%) is not significantly (p > 0.05) different from that among the other castes or the tribal populations of the region (CR: 5–12.9%, 11.2–18.3%, respectively). On the other hand, none of the 159 Brahmins and Kshatriyas from the northern states of India (Punjab, Rajasthan, Uttar Pradesh and West Bengal) belong to M2 while the frequency reaches nearly 3% (CR: 1.6–4.6%) among the other castes and tribal populations of the region. Table 3 Indian-specific sub-clades of mtDNA haplogroups M and R. DIAGNOSTIC CODING REGION MARKERS ANCESTRAL HVS-I MOTIF COALESCENCE (years) ρ/nδ2 a COALESCENCE b (years) ρ/nδ2 a nc Proportion (95% CR) M2 477G-1780-8502 16223–16319 70,600 ± 21,000 0.02 70,100 ± 20,700 0.04 166 6.1 (5.3–7.1)  M2a 5252–8369 16223-16319-16270 48,300 ± 20,100 0.03 46,700 ± 22,800 0.06 79 2.9 (2.3–3.6)  M2b 16223-16319-16274 54,800 ± 25,000 0.02 57,600 ± 22,300 0.06 87 3.2 (2.6–3.9) M3a 4580 16126–16223 17,300 ± 7,400 0.10 17,300 ± 7,600 0.11 62 3.0f (2.4 – 3.8) M4a 6620–7859 16223–16311 19,200 ± 9,000 0.23 19,100 ± 9,000 0.25 21 0.8 (0.5–1.2) M6 3537 16223-16231-16362 33,000 ± 13,900 0.04 30,000 ± 13,600 0.08 79 2.9 (2.3–3.6)  M6a 16223-16231-16356-16362 19,100 ± 7,600 0.17 15,700 ± 8,100 d 0.54 37 1.4 (1.0–1.9)  M6b 5585 16188-16223-16231-16362 6,000 ± 2,100 0.73 12,100 ± 4,500 0,82 37 1.4 (1.0–1.9) M18 16223-16318T 9,400 ± 3,200 0.31 17,100 ± 4,700 0.61 58 2.1 (1.7–2.7) M25 15928 16223–16304 19,400 ± 7,200 0.30 22,300 ± 8,600 0.32 25 1.0g (0.7 – 1.5) R2e 4216 16071 40,400 ± 14,300 0.45 52,500 ± 21,700 0.5 8 0.3 (0.2–0.6) R5 8594 16266–16304 66,100 ± 22,000 0.04 69,800 ± 24,800 0.05 58 2.1 (1.7–2.7) R6 -12282 AluI 16129–16362 30,000 ± 11,000 0.14 30,300 ± 11,600 0.19 35 1.3 (0.9–1.8) Total 2719 a Efficiency of the sample for coalescence calculation (Saillard et al. 2000) b Calculated without the tribal populations c In the Indian subcontinent (a total of 2719 mtDNAs) d Excluding also the scheduled cast of Mukri e R2 is spread also in the Near East, Central Asia and Volga basin f Excluding the datasets where M3* / M3a discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 382 Pushtoon, Koragas, Naga, Yerava, Pardhi, Paniya Adi, Andh, Apatani and Soligas (Cordaux et al. 2003); 35 Kota and Kurumba (Roychoudhury et al. 2001) g Excluding the datasets where M* / M25 discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 39 Thoti and 29 subjects from Bangladesh (Cordaux et al. 2003); 12 Muria (Roychoudhury et al. 2001) Figure 1 The spatial distribution of Indian-specific mtDNA haplogroups (M2, M6 and M3) and their sub-haplogroups. Maps of South and Southwest Asia depicting the spatial frequency distribution of various Indian-specific mtDNA haplogroups. For India, the tribal populations were excluded (see 'Methods' for explanation). The published (full reference in Table 6, see Additional file 1) and new data were averaged to the resolution of states in India, geographic regions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate sample sizes. In the case of haplogroup M3a some datasets had to be excluded because the discrimination between M* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). We found that R5, which is defined by transitions at nps 8594 [27], 16266 and 16304, is the second most frequent sub-clade of R in India after haplogroup U. The coalescence age estimate for R5 was similar to that of M2 (Table 3), whereas individual boughs within the R5 limb showed expansions from ca. 20,000 ybp to ca. 50,000 ybp (Figure 12, see Additional file 8). Our data indicate that this diverse and ancient haplogroup is present over most of India (Figure 3, panel R5). Though absent among the Austro- Asiatic tribal groups, R5 is spread across the other language boundaries. In contrast to M2, R5 is more frequent among the caste (CR: 2.5–4.5%) than among the tribal populations (CR: 0.7–1.9%) (Table 2). Figure 3 The spatial distribution of Indian-specific mtDNA haplogroups (R5 and R6) and West Eurasian-specific U7, W and R2 in South and southwest Asia. For other details see legend to Figure 1. Together with the Indian-specific sub-clades of haplogroup U2 [13,27], haplogroups M2 and R5 can be discriminated as a package of Indian-specific mtDNA clades harboring extremely deep coalescence times (around 50,000 – 70,000 ybp). Together they constitute nearly 15% of the Indian mtDNAs. Importantly, these haplogroups are virtually absent elsewhere in Eurasia [13,15], this study]. Because most of Indian varieties of haplogroup M are still unclassified (M*), this package is likely to be extended when more mtDNA coding region information will become available for the M* lineages in India. The geographic distribution of the M2, R5 and U2i package seems to be rather uniform in the context of the Indian-specific maternal lineages (SAA p > 0.05). When excluding the mtDNAs that are likely to have arrived more recently from West or East Eurasia, the share of the package among the caste populations in northern and southern India is roughly similar (CR: 16.1–24.5% and 19.5–26.0 %, respectively). However, in accordance with expectations from the individual haplogroup distributions, the tribal groups speaking Austro-Asiatic and Tibeto-Burman languages are characterized by considerably lower values (CR: 3.9–14.1% and 3.8–15.1%, respectively). The quest for finding the origin of haplogroup M and a plausible scenario for the peopling of Eurasia Based on the high frequency and diversity of haplogroup M in India and elsewhere in Asia, some authors have suggested (versus [3]) that M may have arisen in Southwest Asia [16,17,31]. Finding M1 or a lineage ancestral to M1 in India, could help to explain the presence of M1 in Africa as a result of a back migration from India. Yet, to date this has not been achieved [15], this study). Therefore, one cannot rule out the still most parsimonious scenario that haplogroup M arose in East Africa [3]. Furthermore, the lack of L3 lineages other than M and N (indeed, L3M and L3N) in India is more consistent with the African launch of haplogroup M. On the other hand, one also observes that: i) M1 is the only variant of haplogroup M found in Africa; ii) M1 has a fairly restricted phylogeography in Africa, barely penetrating into sub-Saharan populations, being found predominantly in association with the Afro-Asiatic linguistic phylum – a finding that appears to be inconsistent with the distribution of sub-clades of haplogroups L3 and L2 that have similar time depths. That, plus the presence of M1 without accompanying L lineages in the Caucasus [32] and [our unpublished data], leaves the question about the origin of haplogroup M still open. In contrast to haplogroup M, ancient sub-clades of haplogroup N are spread both east and west of India as well as within India itself. Several migration scenarios involving multiple "out of Africa" events punctuated by space, time or both, could be invoked to explain the phylogeography of these mtDNA haplogroups. Yet, using the parsimony criterion it can be argued that only a single early migration that brought ancestral lineages, M and N (with the latter having already given rise to R), to South Asia could account for the extant mtDNA phylogeography in Eurasia [15]. The finding of several largely South and West Asian-specific sub-clades (H, L, R2, and F*) of the major Eurasian Y chromosomal haplogroups F and K also supports this scenario [15]. From South and West Asia the colonization would have sprung both east and west as region-specific mtDNA and Y chromosomal sub-clades appeared both in West- and East-Eurasia as well as in India itself (Figure 5). However, not all the West-Eurasian Y-chromosomal founder haplogroups are present in India. Haplogroup E, for example, was possibly carried to Europe and Western Asia later via the Levantine corridor [33]. Similarly, a Late Upper Palaeolithic origin and spread of mtDNA haplogroup X from Northeast Africa and Middle East has been suggested lately [34]. Figure 5 Peopling of Eurasia. Map of Eurasia and northeastern Africa depicting the peopling of Eurasia as inferred from the extant mtDNA phylogeny. The bold black arrow indicates the possible "coastal" route of colonization of Eurasia by anatomically modern humans (ca. 60,000 – 80,000 ybp.). This "Southern Coastal Route" is suggested by the phylogeography of mtDNA haplogroup M, the virtual absence of which in the Near East and Southwest Asia undermines the likelihood of the initial colonization of Eurasia taking a route north around the Red Sea. Therefore, the initial split between West and East Eurasian mtDNAs is postulated between the Indus Valley and Southwest Asia. Spheres depict expansion zones where, after the initial (coastal) peopling of the continent, local branches of the mtDNA tree (haplogroups given in the spheres) arose (ca. 40,000 – 60,000 ybp), and from where they where further carried into the interior of the continent (thinner black arrows). Admixture between the expansion zones has been surprisingly limited ever since. We note that while there is no obvious need to introduce the "northern route" – from northeast Africa over Sinai to the Near East – to explain the initial colonization of Eurasia, the spread of some mtDNA and Y-chromosomal haplogroups implies that the "northern" passage might have been used in a later period [33, 34]. The improved structure of autochthonous Indian mtDNA clades Nearly a third of Indian mtDNAs belonging to haplogroup M could be assigned to its existing boughs and limbs with the current knowledge of the mtDNA coding region polymorphisms (Table 9, see Additional file 4). It is likely that the unclassified Indian M* and R* mtDNAs are also to a large extent autochthonous because neither the East nor West Eurasian mtDNA pools include such lineages at notable frequencies. Haplogroup M6 (Figure 6) is primarily found in the Indus Valley and on the western shores of the Bay of Bengal where its sub-clades M6a and M6b are concentrated towards the southwest and the northeast, respectively (Figure 1, panel M6, M6b cline is significant SAA p < 0.05, Figure 4). The highest frequencies of M6a and M6b were found amongst the Mukri scheduled caste from Karnataka (17%) and in Kashmir (10%), respectively. The Mukri form an endogamous group of no more than 10,000 individuals, who dwell on an area less than 2000 km2 [35]. That, together with the observation that all the sixteen M6 sequences found among the Mukri belong to a single haplotype, suggests that genetic drift has played a major role in the demographic history of the Mukri. The statistical significance of the high M6 frequency in Kashmir is undermined by the small sample size (19 individuals), which results in the very wide error margins for the frequency estimate (CR: 3.2–31.7%). Figure 6 Network of HVS-I haplotypes belonging to haplogroup M6. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. Character changes are specified only for transversions. Underlined samples are those in which the diagnostic coding region markers (3537 and 5585) were assayed by either RFLP analysis (M6: -3537 AluI; M6b: -5584 AluI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). The coalescence estimate marked with two asterisks is calculated without the data on tribal and scheduled caste (the Mukri) populations (see text for details). Different geographic distributions characterize the sub-clades of M3 and M4 that we define by mtDNA coding region markers (Table 3, Figures 7 and 8). Both M3a and M4a show time depths around 20,000 ybp. However, while M4a is sparsely spread in most of India with no obvious geographical cline (Figure 2, panel M4a, SAA p > 0.05), the spread of M3a is concentrated into northwestern India (Figure 1, panel M3a, SAA p < 0.05), suggesting that the region may have been the ancestral source. The frequency of M3a is at its highest amongst the Parsees of Mumbai (22%). Given the low M3a diversity amongst the Parsees – the twelve M3a mtDNAs fall into the two most common haplotypes (Figure 7) – the high frequency is likely a result of admixture and subsequent founder events. On the other hand, it is intriguing that, despite its low frequency, M3a penetrates into central and southwestern Iran (Figure 1, panel M3a) – the historic origin of the Zoroastrian Parsees. In addition to the Parsees we found M3a at high frequencies amongst the Brahmins of Uttar Pradesh (16%) and the Rajputs of Rajasthan (14%) (Table 10, see Additional file 5). Figure 7 A network of haplogroup M3a haplotypes. Underlined samples are those in which the diagnostic coding region marker (4580) of M3a was assayed by either RFLP analysis (-4577 NlaIII) or direct sequencing. For other details, see the legend to Figure 6. Figure 8 Network of HVS-I haplotypes belonging to haplogroup M4a. Underlined samples are those in which the diagnostic coding region marker (6620) of M4a was assayed by either RFLP analysis (+6618 MboI) or direct sequencing. For other details, see the legend to Figure 6. Awaiting further information from complete mtDNA sequences, we defined haplogroup M18 by using the transversion at np 16318. This star-like cluster (Figure 9) is spread at low frequencies across India, with the exceptions of the very north and the coast of the Arabian Sea (Figure 2, panel M18). The high incidence (33%) of the M18 nodal haplotype among the Austro-Asiatic speaking Lodha of West Bengal (Table 10, see Additional file 5) suggests a possible founder effect in this population. This explains the nearly two-fold difference between the coalescence estimates for this cluster calculated with and without the tribal data (Table 3). Figure 9 Network of HVS-I haplotypes belonging to haplogroup M18. For other details, see the legend to Figure 6 The G to A transition at np 15928 has been spotted on different branches (e. g. haplogroups T and M) of the mtDNA phylogeny [3,36]. Quintana-Murci and colleagues observed this transition within haplogroup M in combination with the HVS-I motif 16048-16129-16223-16390 [27]. None of the mtDNAs in our study which harbor – or stem from – the 16048-16129-16223 motif were positive for the 15928 transition, suggesting an additional occurrence. In addition, we recorded this transition associated with three other HVS-I motifs on the background of haplogroup M (M8-Z: 16185-16223-16260-16298; M*: 16223 and M*: 16086-16223-16335). These occurrences cannot be monophyletic for obvious reasons. Yet, when combined with the transition at np 16304, G15928A roots a star-like subclade of haplogroup M that we tentatively named M25 (Figure 10). In this case, monophylecity is the most parsimonious assumption. This haplogroup is moderately frequent in Kerala and Maharashtra but rather infrequent elsewhere in India (Figure 2, panel M25). Figure 10 Network of HVS-I haplotypes belonging to haplogroup M25. Underlined samples are those in which the diagnostic coding region marker (15928) of M25 was assayed by either RFLP analysis (-15925 MspI) or direct sequencing. For other details, see the legend to Figure 6. Coalescence estimates for these Indian-specific mtDNA haplogroups (M3a, M4a, M6, M25 and R6) fall largely between 20,000 and 30,000 ybp. These estimates overlap with those of many West Eurasian-specific (e.g. H, HV, preHV, U3, U4, K, X [9,34]) and East Eurasian-specific (A, F2, D4, M7c1, M7a1, M8a [7,22]) mtDNA clades, suggesting a rather synchronic worldwide demographic expansion event in the late Pleistocene, during an interglacial period preceding the LGM. Several Indian-specific mtDNA clades demonstrate a similar spread-pattern in southern India. We found haplogroups M4a, M6a and M18 in southeastern Tamil Nadu and Andhra Pradesh while they were absent from neighboring Karnataka and Kerala (Figure 1 panel M6a and Figure 2 panels M4a and M18). One possible explanation is that admixture has been facilitated along the coastlines of the Arabian Sea and the Bay of Bengal. On the other hand, because the absolute frequencies of these haplogroups are rather low, it cannot be ruled out that an increase of sample sizes would disrupt the observed spread-pattern. Were the Austro-Asiatic speaking tribal people the earliest inhabitants of India? By calculating nucleotide diversities and expansion times (using the method from [37]) for different linguistic groups of India, some previous studies on mtDNA variation have distinguished the Austro-Asiatic speaking tribal groups as the carriers of the genetic legacy of the earliest settlers of the subcontinent [17,38]. However, because the linguistic groups of India do not cluster into distinct branches of the Indian mtDNA tree [13,15,19], this study], calculating the beginning of expansion for those groupings is problematic and likely controversial as well. Recently, Basu et al. (2003) supported the conclusions of [17,38] by reporting that the frequency of the ancient haplogroup M2 among the Austro-Asiatic tribal populations is as high as 19%, and that they lack the slightly younger haplogroup M4 (the likely paraphyletic mother-clade of M4a). The authors have regarded the HVS-I transition at np 16319 as sufficient in defining haplogroup M2. This assumption, however, might lead to an overestimation of M2 frequency and age. Indeed, the 16319 transition has arisen several times on the background of other Indian haplogroup M lineages (Table 7, see Additional file 2), more specifically, in mtDNAs lacking the coding region markers that define M2 [15]. Although two out of the four M2 sequences reported by Basu et al. (2003) among the Lodha, Mundas and Santals (HVS-I sequences originally published by [17]) do harbor the characteristic M2a HVS-I motif (16223-16270-16274-16319-16352), without information from the coding region it is not clear whether the other two sequences (HVS-I motifs: 16092-16179-16223-16289-16294-16319 and 16147G-16172-16223-16319) represent novel M2 sub-clades (because these sequences cannot be affiliated with M2a or M2b) or derive from two independent branches of haplogroup M where 16319 transition has arisen recurrently. HVS-I motif 16147G-16172-16223, for example, is commonly associated with haplogroup N1a. Since sequence data on the five M2s among the Austro-Asiatic speaking tribe Ho, reported by Basu et al. (2003), have not been made available in the publication, we cannot rely on their haplogroup classification. Thus, we are left with one Munda and one Santal mtDNA belonging to haplogroup M2. They make up just 5% of the Austro-Asiatic tribal sample of 37 subjects (excluding the ten Ho). Interestingly, we found no instances of haplogroup M2 among the 56 Lodhas analyzed in this study. Consequently, when excluding the recurrences of the 16319 transition on the background of other sequence motifs, the frequency of M2 among the Austro-Asiatic speaking tribal groups from West Bengal in the combined dataset (Table 7, see Additional file 2) is significantly reduced to about 2%. The corrected value is comparable to the M2 frequency (>3%) in tribal populations speaking Indo-European languages of Punjab and Uttar Pradesh, but is significantly lower than its frequency (>14%) among the Dravidic-speaking tribal groups of Andhra Pradesh (Table 8, see Additional file 3). Language families present today in India, such as Indo-European, Dravidic and Austro-Asiatic, are all much younger than the majority of indigenous mtDNA lineages found among their present-day speakers at high frequencies (see Additional file 9). It would make it highly speculative to infer, from the extant mtDNA pools of their speakers, whether one of the listed above linguistically defined group in India should be considered more "autochthonous" than any other in respect of its presence in the subcontinent. Additionally, we note that some recent linguistic and archaeological evidence place the spread of the Austro-Asiatic languages in the Neolithic, in conjunction with the dispersal of rice cultivation from the Yangtze River basin [39]. If this were the case, it would imply that the arrival of this linguistic phylum in India was not associated with female gene flow. Gene flow from West Eurasia Broadly, the average proportion of mtDNAs from West Eurasia among Indian caste populations is 17% (Table 2). In the western States of India and in Pakistan their share is greater, reaching over 30% in Kashmir and Gujarat, nearly 40% in Indian Punjab, and peaking, expectedly, at approximately 50% in Pakistan (Table 11, see Additional file 6, Figure 11, panel A). These frequencies demonstrate a general decline (SAA p < 0.05 Figure 4) towards the south (23%, 11% and 15% in Maharashtra, Kerala and Sri Lanka, respectively) and even more so towards the east of India (13% in Uttar Pradesh and around 7% in West Bengal and Bangladesh). The low (<3%) frequency of the western Eurasian mtDNAs in Rajasthan may be in part a statistical artifact due to the limited sample size of 35 Rajputs. Figure 11 The segregation of West Eurasian, East Eurasian and South Asian mtDNA pools. Partial map of Eurasia illustrating the spatial frequency distribution of mtDNA haplogroups native to West Eurasia (panel A), South Asia (panel B) and East Eurasia (panel C). Data points represent states in India, geographic divisions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate the applicable sample size. The distribution of West Eurasian-, South Asian- and East Eurasian-specific mtDNA haplogroups amongst the tribal populations of South and Southeast Asia are depicted as pie diagrams on panel D (colors as on panels A, B, and C, while the white color represents unclassified M* and R* mtDNAs). The data on tribal populations was not used for the isofrequency maps of panels A, B and C (see Materials and Methods for explanation). In comparison to an overall frequency of 17% (CR 15.1–19.3%) among the caste populations, only 7% (CR 6.2 – 9.0%) of the mtDNAs from the tribal groups show affiliation to the West Eurasian haplogroups. The observed difference could be caused by differences in the extent of gene flow from the west to different social layers of the Indian society [19], and/or a more pronounced genetic drift among the tribal groups. This western Eurasian contribution into the Indian maternal gene pool can be broadly divided into two different components. Over two thirds of the West Eurasian-specific mtDNAs found in India are made up by haplogroups HV, TJ, N1 and West Eurasian-specific branches of haplogroup U. It is likely that these mtDNA haplogroups have been carried to western India both by relatively low-intensity long-lasting admixture at the border regions as well as a consequence of numerous but probably limited migrations during the last 10, 000 ybp [13]. The remaining one third of the West Eurasian-specific mtDNAs found in India is comprised of haplogroups U7, R2 and W showing much deeper time depths in India – approximately forty thousand years before present (Table 4). A large-scale immigration – carrying these haplogroups – could have introduced a substantial fraction of the diversity already present within the putative source areas. This would explain the deep coalescence times of these haplogroups in India, while their actual arrival could have occurred later. Alternatively, the coalescence estimates may indeed reflect a deeper autochthonous history of these mtDNA clades in India. It is worthwhile to stress that while in India the share of U7, R2 and W in the West Eurasian-specific mtDNAs mounts to nearly a third, in Iran it stays below 15%. Table 4 Coalescence estimates and diversity values for mtDNA haplogroups U7, W and R2 in India, Central Asia, Near and Middle East. INDIA NEAR AND MIDDLE EAST CENTRAL ASIA HAPLO-GROUP COALESCENCE (years) DIVERSITY COALESCENCE (years) DIVERSITY COALESCENCE (years) DIVERSITY U7 41,400 ± 15,800 0.941 41,200 ± 14,800 0.91 34,400 ± 15,500 0.941 W 37,900 ± 11,100 0.883 32,000 ± 8,700 0.934 27,400 ± 8,300 0.758 R2 40,400 ± 14,300 0.923 36,100 ± 11,100 0.955 25,200 ± 13,300 0.889 Similarly to HV, TJ, N1, the spread of U7, W and R2 in India is geographically uneven – the three haplogroups are much more frequent in the northwestern states (SAA p < 0.05, Figure 4). Together, they constitute nearly 14% of the maternal gene pool in Indian Punjab, Gujarat and Rajasthan. Their frequency is also high in Iran (13%) and Pakistan (10%) but declines in Central Asia (5%). Although haplogroup W is not highly frequent in European populations, it is nevertheless quite common [9], reaching the highest frequencies among the central-northern Finns (9% [40]). Yet, it is virtually absent from the Finno-Ugric speaking populations of the Volga basin [41]. In Central Asia the frequency of haplogroup W stays below 2%. Many European populations are lacking haplogroup U7 [9], but its frequency climbs over 4% in the Near East and up to 5% in Pakistan, reaching nearly 10% level in Iranians (Table 9, see Additional file 4). In India, haplogroup U7 frequency peaks at over 12% in Gujarat, in the westernmost state of India, while for the whole of India its frequency stays around 2%. Expansion times and haplotype diversities for the Indian and Near and Middle Eastern U7 mtDNAs are strikingly similar (Table 4). The possible homeland of this haplogroup spans likely in Gujarat and Iran because from there its frequency declines steeply both to the east and to the west. If the origin were in Iran rather than in India, then its equally high frequency as well as diversity in Gujarat favors a scenario whereby U7 has been introduced to the coastal western India either very early, or by multiple founders. Notably, the overlap of the Indian and Iranian lineages is largely restricted to the ancestral nodes while the coalescence age estimates for the nearly exclusively Indian (16207-16309-16318T) and West Eurasian (16126-16309-16318T) founder HVS-I motifs both yield time depths of about 20,000 to 30,000 ybp. Haplogroup R2 appears at low frequencies in Near and Middle East and India and is virtually absent elsewhere. The spread of haplogroup R2 in Europe is restricted to a few populations in the Volga basin where it is represented by nodal haplotypes and by a region-specific subclade characterized by the HVS-I motif 16037–16172 [41]. The coalescence estimate of this sub-clade is 11,400 ± 9,000 ybp. However, its wide error range prevents us from drawing any firm conclusions. To summarize, the West and South Asian phylogeography of haplogroups W, U7 and R2 can be viewed as a genetic continuum that spans from the Near East into India, extending north into Central Asia. The coalescence times of these haplogroups suggest that this continuum took shape somewhere between 30,000 to 50,000 ybp (Table 4), thus falling within the climatically favorable interglacial period. We notice that the extant U7 and W frequencies along the proposed continuum are not uniform. U7 is more predominant in Iran, Pakistan, northwestern India and the Arabian peninsula, while W is more frequent in the western Near-East, Anatolia and the Caucasus. The coalescence ages of the Indian- and Iranian-specific U7 clades suggest that the time-window of this continuum was closed by ca. 20,000 ybp. The inferred extreme aridity of eastern Iran and western India during the last glacial maximum, which is well documented in paleovegetation reconstructions [42] may explain the observed segregation. It has been suggested that the Jews settled in southwest India on the coast of the Arabian Sea sometime during the early Middle Ages. However, the mtDNA pool of the extant Cochin Jews is overwhelmingly Indian-specific (Table 10, see Additional file 5). We found exact or close matches to the fourteen HVS-I haplotypes observed among the Cochin Jews in other Indian populations. It is not clear whether the Near Eastern mtDNA lineages have been lost or the initial Jewish settlers did not include women. Gene flow from East Eurasia The East Eurasian-specific mtDNA haplogroups are less common in India and more sharply geographically segregated than the haplogroups of western Eurasian ancestry (Table 2; Figure 11, panel C). Indian caste populations harbor only about 4% of such mtDNAs, compared to 17% of the West Eurasian ones (Table 2). Elevated frequencies of haplogroups common in eastern Eurasia are observed in Bangladesh (17%) and Indian Kashmir (21%) and may be explained by admixture with the adjacent populations of Tibet and Myanmar (and possibly further east: from China and perhaps Thailand). On the other hand, the high frequencies of East Eurasian-specific mtDNAs found in the southern Indian state of Tamil Nadu (21%) are unexpected when considering their relatively low frequencies (~1%) in West Bengal and Andhra Pradesh. We notice, however, that the haplogroup assignments used here for the Tamil Nadu sample (A4, B4, F1a and M7) (Table 7, see Additional file 2) are based on HVS-I sequences alone [29]. As shown and discussed elsewhere [7], such type of assignment is prone to mistakes. Two varieties of haplogroup M, D4c and G2a, were recently identified as largely specific to Central Asia [43]. In spite of geographical proximity we did not find these haplogroups in northern or northwestern India. Haplogroup G2a did, though in marginally low frequency, turn up in Iran (CR: 0.1 – 1.6%) and in southern Indian states Andhra Pradesh (CR: 0.3 – 2.9%) and Sri Lanka (CR: 0.2 – 4.1%). Tibeto-Burman speaking tribal populations of eastern and northern India exhibit the highest frequencies of East Eurasian-specific mtDNA haplogroups. As inferred from the published HVS-I sequences [29], their share sums up to approximately two thirds of mtDNAs among the tribal groups in Assam, Nagaland, Arunachal Pradesh and Tripura, (Table 8, see Additional file 3; Figure 11, panel D). MtDNA haplogroups native to East Eurasia are also highly frequent in the northern states of India, reaching a peak of nearly 50% among the Kanet of Himachal Pradesh. Papiha and colleagues have previously demonstrated through the typing of immunoglobin allotypes that the Tibetan admixture among the regional Kanet groups decreases as the distance from the Tibetan border increases [44]. Thus, mtDNA data are consistent with an ancestral origin of the Tibeto-Burman speaking tribal populations outside (east of) India in the neighboring Tibet and Myanmar [30,45]. Haplotype sharing between populations The majority (70%) of the 1136 mtDNA haplotypes found among continental Indians (including Pakistan and Bangladesh) are singletons and 41% of those that occur more than once are restricted to a single population. Only a few haplotypes are shared among five or more populations. The number of shared haplotypes between pairs of social, linguistic and geographic groups of Indian populations is slightly (but in most cases insignificantly) lower than that between random groups of Indian populations taken for reference (see Materials and Methods). Where the decline of shared haplotypes is significant relative to the reference, it is most probably caused by large differences in the sample sizes of the groups under comparison (Table 5). Table 5 Proportion of mtDNA haplotype sharing between population groups of South Asia Group 1 Group 2 pop. group n nb 1/2d pop. group n nb 2/1e nc Random 1g 1256 ± 215 617 ± 109 .399 – .453 Random 2g 1426 ± 215 643 ± 114 .396 – .447 132 (112 – 154) Tribals 1197 437 .411 – .467 Castes 1485 806 .324 – .372 107 (90 – 128) Northern states 1204 647 .277 – .329 Southern states 1478 585 .345 – .394 96 (80 – 116) Western states 1142 508 .453 – .511 Eastern states 1540 735 .331 – .379 107 (90 – 128) Dravidians 974 380 .428 – 490 Others 1708 846 .273 – .317 90 (74 – 109) Indo-Europeans 1322 686 .328 – .379 Others 1360 556 .393 – .446 106 (89 – 127) AA tribals 90 35 .552 – .746 Others 2592 1110 .014 – .025 9 (5 – 17) TB tribals 249 142 .174 – .277 Others 2433 1018 .091 – .115 24 (16 – 35) Indo-Europeans 1322 686 .295 – .346 Dravidians 974 380 .425 – .487 87 (71 – 106) Total 2682 1136 340 f a continental Indians including Pakistan and Bangladesh (excluding Andaman & Nicobar) b number of haplotypes c number of shared haplotypes between group 1 and group 2 (95% credible regions; CR) d CR of the proportion of those haplotypes in group 1 that occur also in group 2 e CR of the proportion of those haplotypes in group 2 that occur also in group 1 f 41% of the shared haplotypes are shared only between members of the same population g ± indicate SD An alternative method that assesses the degree of haplotype sharing between populations is to investigate the combined frequency of the shared haplotypes in two population groups. Thus, amongst the northern and the southern population groups the combined frequency of the haplotypes present also in the other group is significantly lower than that which we observed in the case of random groups. This is not surprising because West Eurasian-specific mtDNA haplogroups are rather frequent in northwest India. Because the Indo-European and the Dravidic speakers of India are largely concentrated to the northern and southern parts of the subcontinent, respectively, the differences arising from geographic division of the Indian populations also correspond to these linguistic groupings (Table 5). Conclusions Three Indian-specific haplogroups, M2, U2i and R5, which encompass about 15% of the Indian mtDNA pool, exhibit equally deep coalescence ages of about 50,000 – 70,000 years. Thus, their spread can be associated with the initial peopling of South Asia. Haplogroups U7, W and R2 harbor a number of similar traits. Their overlapping geographic distributions and coalescence times suggest some degree of genetic continuum in the area spanning from the Near and Middle East through northwest India and reaching north into Central Asia. The coalescence estimates for these haplogroups are equally deep (around 30,000 – 50,000 years) in these different regions. That may be a result of either relatively more recent albeit large in scale migrations that brought along most of the diversity or may indeed reflect the region-specific expansions of these haplogroups. The former explanation could be ruled out since it is impossible to envisage a substantial movement of mtDNAs from South Asia that would not include haplogroup M. The same is true for the opposite – the share of U7, W and R2 within the West Eurasian-specific mtDNA haplogroups is two-fold higher in India than it is in Iran. Moreover, the South- and West Asian-specific sub-branches of haplogroup U7 predate the last glacial maximum. Therefore, deep autochthonous history of these haplogroups in the region remains to be the most parsimonious explanation. Through the use of mtDNA coding region markers, we were able to classify altogether a quarter of the Indian M and R mtDNAs into a number of Indian-specific mtDNA haplogroups, four of which we newly identified. Several of these are characterized by clear patterns in their geographic distribution and/or different frequencies among different socio-cultural groups of India. Additional efforts should be undertaken to identify new coding region markers in order to further improve the classification of Indian mtDNAs. Indeed, much of the information is still hidden by the poor resolution of the Indian mtDNA tree and further piling of HVS-I datasets would add little to deepen our understanding of the demographic history of South and Southwest Asia. We found that haplogroup M frequency drops abruptly from about 60% in India to about 5% in Iran, marking the western border of the haplogroup M distribution. A similarly sharp border cuts the distribution of Indian-specific mtDNA haplogroups to the east and to the north of the subcontinent. We therefore propose that the initial mtDNA pool established upon the peopling of South Asia has not been replaced but has rather been reshaped in situ by major demographic episodes in the past and garnished by relatively minor events of gene flow both from the West and the East during more recent chapters of the demographic history in the region. Methods Subjects MtDNA sequence variation in a total of 796 Indian samples most of which are held in a collection at Newcastle University [46] was analyzed. The samples cover a wide geographical range that spans from Himachal Pradesh in the north, Sri Lanka in the south, West Bengal in the east and Gujarat in the west (Table 1). Tribal populations constitute 15% of the total sample size. The Lodha (n = 56) live mostly in the western part of Midnapore district of West Bengal where they are also known as Kheria or Kharia. Their total population size was ~59000 according to the 1981 census. Their language belongs to the Mundari branch of the Austro-Asiatic language family [47]. The Kanet (n = 37) make up two thirds of the ~50000 inhabitants of the Kinnaur district in Himachal Pradesh [48]. Their language belongs to the Himalayan group of the Tibeto-Burman language family [47]. Five Bhoksa and twenty-six Tharu individuals were included in the present study in addition to those from the same populations that we have previously reported [13]. Most of the Tharu live in the Terai areas (a belt of marshy land at the foot of the Himalayas) of Nepal (n = 720000). Approximately 96000 reside in Uttar Pradesh and Uttaranchal, Indian states adjacent to Nepal. Around 32000 Bhoksas live in the lowland areas of Uttaranchal and the neighboring Bijnor district of Uttar Pradesh. Both these tribal groups speak languages belonging to the Indo-European phylum [47]. The non-tribal Indian samples analyzed contained 105 West Bengalis of different caste rank, 58 Konkanastha Brahmins from Bombay, 53 Gujaratis, 50 Moors and 82 Sinhalese from Sri Lanka, 109 Punjabis of different caste rank from the Punjab, 25 Brahmins from Uttar Pradesh, 35 Rajputs from Rajasthan, 55 Parsees from Maharashtra and 100 subjects from Cochin, Kerala (including 45 Jews who have moved to Israel) (Table 1). The Iranian sample of 436 individuals was collected in different locations mainly from southwestern and northwestern Iran (Table 1). The new Indian mtDNA sequence data was combined with that previously published on Indian populations [3,13,15,17,18,29,35,49-51] to produce a pooled dataset (n = 2572) in which the tribal populations constitute slightly over 50% (Table 6, see Additional file 1). By including also the data on Iranian and published data on Pakistani [13,27,29], Bangladeshi [29], Chinese [7,22,52-54] and Thai [53,55,56] populations (n = 145, 29, 919 and 552, respectively), a dataset of over 4600 samples spanning from West to East Asia (Table 6 and 7, see Additional files 1 and 2) was obtained. In many cases the published data was reanalyzed. Analogy with the newly obtained HVS-I motifs, that were classified into haplogroups using diagnostic markers of the mtDNA coding region, served as basis for haplogroup assignments of the published HVS-I sequences. An even wider range of data was used for the production of the isofrequency maps: the published data from Armenia (n = 192) [32] and Kyrgyz (n = 95) [57], and the unpublished data from Yemen (n = 118), Oman (n = 79), Saudi Arabia (n = 205), Ethiopia (n = 270), Uzbekistan (n = 114) and Tajikistan (n = 41). Further details about the mtDNA variation of these populations will be published elsewhere. MtDNA molecular analyses DNA was extracted using standard phenol-chloroform methods [58]. The hypervariable segment (HVS)-I (between nucleotide positions (np) 16024–16400) of the control region was sequenced in all the 796 Indian and 436 Iranian samples. Preparation of sequencing templates was carried out following Kaessmann et al. [59]. Purified products were sequenced with the DYEnamic™ ET terminator cycle sequencing kit (Amersham Pharmacia Biotech) and analyzed on ABI 377 DNA or Megabace2000 Sequencers. Sequences were aligned and analyzed with the Wisconsin Package (GCG). In addition, informative mtDNA coding region positions [3,11,27] were assayed (Table 7, see Additional file 2) in selected individuals from different HVS-I haplotypes to determine haplogroup affiliations. Data analysis Median networks [60,61] were constructed using Network 3.111 and Network 2.10B programs [62] with default settings (r = 2; ε = 0). MtDNA coding region markers were given five times the weight of the HVS-I positions. Coalescence of mtDNA haplogroups and sub-haplogroups was calculated using ρ (the averaged distance to a specified founder haplotype) and a mutation rate of one transition per 20,180 years between nps 16090–16365 [63]. Standard errors for coalescence estimates and efficiency of a sample for coalescence time calculation (ρ/nδ2) were calculated following [64]. Coalescence times were further calculated with the exclusion of tribal populations sequences, yet preserving the cluster topology (coalescence times marked with an asterisk). A more intense genetic drift (particularly founder effects) could introduce a bias into the coalescence time calculation (for example, see below the coalescence time of haplogroup M18 with and without the Lodha sample). The software kindly provided by Vincent Macaulay was adopted in order to calculate the 95% credible regions (CR) from the posterior distribution of the proportion of a haplogroup/sub-haplogroup in the population. Haplotype diversity was estimated as where n is the number of sequences, k the number of distinct haplotypes, and ni number of sequences with one distinct haplotype. Haplogroup isofrequency maps were generated using the Kriging method in Surfer 7 program of Golden Software. Haplogroup frequencies were averaged over populations from the same state in India, provinces in China and Thailand and geographic divisions in Iran. The data points for Kriging are shown as black dots, while the sample size applicable to the data point is given adjacent to the dot. In relatively small and isolated groups (e.g. tribal groups) random genetic drift might seriously affect the haplogroup frequencies, which may become uninformative when a whole region (e.g. state) is considered (e.g. M18 among the Lodha, see below). Therefore, the tribal data were excluded from the haplogroup isofrequency maps calculation. When illustrating the spread of mtDNA haplogroups native to West Eurasia, East Eurasia and India (Figure 11, panel D) we present these data as pie diagrams. The respective sample size and origin are indicated adjacent to the diagrams. Spatial autocorrelation analysis was done using the PASSAGE software packet [65,66]. The correlograms of Moran's I autocorrelation coefficient were calculated using binary weight matrix with five distance classes. Data on tribal populations was not used (see previous paragraph for explanation). Haplogroup frequencies based admixture proportions were calculated using the ADMIX 2.0 software [67]. Haplotypes were defined as the HVS-I motif combined with the haplogroup label for haplotype analysis. In this way it was possible to discriminate between mtDNAs with identical HVS-I motifs but otherwise known to belong to different haplogroups (e.g. sequences with the CRS motif in HVS-I belonging to haplogroups H or R). In order to assess the results of haplotype sharing analysis between population groups (e.g. tribal and caste populations) we divided the Indian populations ten times randomly into two sets and analyzed the level of haplotype sharing between these sets. Authors' contributions SM and SSP collected most of the Indian samples (in 1970's) and have been keeping the collection in Newcastle University, United Kingdom. MM, TK, GH and KK carried out the RFLP analysis and mtDNA sequencing of the majority of the Indian samples. EM, JP, PS and MK carried out the RFLP analysis and mtDNA sequencing of the Iranian samples. DMB collected the DNA and analyzed mtDNA variation amongst the Indian samples from Cochin and the Cochin Jews collected (the latter) in Israel under supervision of KS, who also contributed to the editing of the manuscript. AT provided some critical new information and, in addition to KS, MTPG and PE, engaged into valuable discussions during manuscript preparation. MM did most of the phylogenetic analysis and was in charge of manuscript writing while TK and RV contributed significantly during both stages and were responsible for conceiving and designing the study. All authors read and approved the final manuscript. Supplementary Material Additional File 3 Table 8. Excel spreadsheet. MtDNA haplogroup frequencies among tribal populations. Frequencies of mtDNA haplogroups amongst the tribal populations of India, China and Thailand as averaged over states of India and provinces in China and Thailand. Click here for file Additional File 7 Table 12. Excel spreadsheet. Comparison of the mtDNA haplogroup frequencies amongst the Indian caste and tribal populations. Click here for file Additional File 4 Table 9. Excel spreadsheet. MtDNA haplogroup frequencies in India and Iran. Click here for file Additional File 6 Table 11. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian caste populations as averaged over states of India. Click here for file Additional File 8 Figure 12. Image file in PNG format. Network of HVS-I haplotypes belonging to haplogroup R5. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. The diagnostic R5 coding region marker 8594 is shown in bold and numbered as in (Anderson et al. 1981). Character changes are specified only for transversions. Underlined samples are those in which the marker 8594 was assayed by either RFLP analysis (-8592 MboI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). Click here for file Additional File 5 Table 10. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian populations. Click here for file Additional File 2 Table 7. Excel spreadsheet. MtDNA variation in the studied populations (raw data). MtDNA control and coding region variation in the populations that were used in the study. The database includes both the newly obtained datasets and the previously published datasets. The latter were in many cases reanalyzed both in silico and by typing for additional mtDNA coding region markers. Click here for file Additional File 1 Table 6. Excel spreadsheet. The list of studied populations. List and details of the populations whose mtDNA were used in the study. This includes both newly obtained datasets and previously published datasets Click here for file Additional File 9 Table 13. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst different linguistic groupings of Indian populations. Click here for file Acknowledgments We thank Lluis Quintana-Murci for providing data prior to publication, Vincent Macaulay for the algorithm for calculating credible regions for haplogroup frequencies, and Jaan Lind and Hille Hilpus for technical assistance. This work was supported by Estonian Science Foundation grants 514 (to RV), 5574 (to TK), 5807 (to EM), the Italian Ministry of the University: Progetti Ricerca Interesse Nazionale 2002 and 2003 (to AT), and European Commission grants ICA1CT20070006 and QLG2-CT-2002-90455 (to RV). ==== Refs Torroni A Miller JA Moore LG Zamudio S Zhuang J Droma T Wallace DC Mitochondrial DNA analysis in Tibet: implications for the origin of the Tibetan population and its adaptation to high altitude Am J Phys Anthropol 1994 93 189 199 8147435 Chen YS Torroni A Excoffier L Santachiara-Benerecetti AS Wallace DC Analysis of mtDNA variation in African populations reveals the most ancient of all human continent-specific haplogroups Am J Hum Genet 1995 57 133 149 7611282 Quintana-Murci L Semino O Bandelt H-J Passarino G McElreavey K Santachiara-Benerecetti AS Genetic evidence of an early exit of Homo sapiens sapiens from Africa through eastern Africa Nat Genet 1999 23 437 441 10581031 10.1038/70550 Alves-Silva J da Silva Santos M Guimarães PE Ferreira AC Bandelt H-J Pena SD Prado VF The ancestry of Brazilian mtDNA lineages Am J Hum Genet 2000 67 444 461 10873790 10.1086/303004 Torroni A Schurr TG Cabell MF Brown MD Neel JV Larsen M Smith DG Vullo CM Wallace DC Asian affinities and continental radiation of the four founding Native American mtDNAs Am J Hum Genet 1993 53 563 590 7688932 Schurr TG Sukernik RI Starikovskaya YB Wallace DC Mitochondrial DNA variation in Koryaks and Itel'men: population replacement in the Okhotsk Sea-Bering Sea region during the Neolithic Am J Phys Anthropol 1999 108 1 39 9915299 10.1002/(SICI)1096-8644(199901)108:1<1::AID-AJPA1>3.0.CO;2-1 Kivisild T Tolk H-V Parik J Wang Y Papiha SS Bandelt H-J Villems R The emerging limbs and twigs of the East Asian mtDNA tree Mol Biol Evol 2002 19 1737 1751 12270900 Kivisild T Kaldma K Metspalu M Parik J Papiha SS Villems R Papiha SS, Deka R, Chakraborty R The place of the Indian mitochondrial DNA variants in the global network of maternal lineages and the peopling of the Old World Genomic Diversity 1999 Kluwer Academic/Plenum Publishers 135 152 Richards M Macaulay V Hickey E Vega E Sykes B Guida V Rengo C Sellitto D Cruciani F Kivisild T Tracing European founder lineages in the Near Eastern mtDNA pool Am J Hum Genet 2000 67 1251 1276 11032788 Derbeneva OA Starikovskaia EB Volod'ko NV Wallace DC Sukernik RI [Mitochondrial DNA variation in Kets and Nganasans and the early peoples of Northern Eurasia] Genetika 2002 38 1554 1560 12500682 Macaulay VA Richards MB Hickey E Vega E Cruciani F Guida V Scozzari R Bonné-Tamir B Sykes B Torroni A The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs Am J Hum Genet 1999 64 232 249 9915963 10.1086/302204 Ballinger SW Schurr TG Torroni A Gan YY Hodge JA Hassan K Chen KH Wallace DC Southeast Asian mitochondrial DNA analysis reveals genetic continuity of ancient mongoloid migrations Genetics 1992 130 139 152 1346259 Kivisild T Bamshad MJ Kaldma K Metspalu M Metspalu E Reidla M Laos S Parik J Watkins WS Dixon ME Deep common ancestry of Indian and western-Eurasian mitochondrial DNA lineages Curr Biol 1999 9 1331 1334 10574762 10.1016/S0960-9822(00)80057-3 Kivisild T Papiha SS Rootsi S Parik J Kaldma K Reidla M Laos S Metspalu M Pielberg G Adojaan M Renfrew C, Boyle K An Indian Ancestry: a key for understanding human diversity in Europe and beyond Archaeogenetics: DNA and the population prehistory of Europe 2000 Cambridge: McDonald Institute for Archaeological Research University of Cambridge 267 279 Kivisild T Rootsi S Metspalu M Mastana S Kaldma K Parik J Metspalu E Adojaan M Tolk H-V Stepanov V The genetic heritage of the earliest settlers persists both in Indian tribal and caste populations Am J Hum Genet 2003 72 313 332 12536373 10.1086/346068 Roychoudhury S Roy S Dey B Chakraborty M Roy M Roy B Ramesh A Prabhakaran N Rani MVU Vishwanathan HMM Fundamental genomic unity of ethnic India is revealed by analysis of mitochondrial DNA Current Science 2000 79 1182 1192 Roychoudhury S Roy S Basu A Banerjee R Vishwanathan H Usha Rani MV Sil SK Mitra M Majumder PP Genomic structures and population histories of linguistically distinct tribal groups of India Hum Genet 2001 109 339 350 11702215 10.1007/s004390100577 Bamshad MJ Watkins WS Dixon ME Jorde LB Rao BB Naidu JM Prasad BV Rasanayagam A Hammer MF Female gene flow stratifies Hindu castes Nature 1998 395 651 652 9790184 10.1038/27103 Bamshad M Kivisild T Watkins WS Dixon ME Ricker CE Rao BB Naidu JM Prasad BV Reddy PG Rasanayagam A Genetic evidence on the origins of Indian caste populations Genome Res 2001 11 994 1004 11381027 10.1101/gr.GR-1733RR Passarino G Semino O Bernini LF Santachiara-Benerecetti AS Pre-Caucasoid and Caucasoid genetic features of the Indian population, revealed by mtDNA polymorphisms Am J Hum Genet 1996 59 927 934 8808610 Basu A Mukherjee N Roy S Sengupta S Banerjee S Chakraborty M Dey B Roy M Roy B Bhattacharyya N Ethnic India: a genomic view, with special reference to peopling and structure Genome Res 2003 13 2277 2290 14525929 10.1101/gr.1413403 Yao YG Kong QP Bandelt HJ Kivisild T Zhang YP Phylogeographic differentiation of mitochondrial DNA in Han Chinese Am J Hum Genet 2002 70 635 651 11836649 10.1086/338999 Kong QP Yao YG Sun C Bandelt HJ Zhu CL Zhang YP Phylogeny of East Asian mitochondrial DNA lineages inferred from complete sequences Am J Hum Genet 2003 73 671 676 12870132 10.1086/377718 Passarino G Semino O Quintana-Murci L Excoffier L Hammer M Santachiara-Benerecetti AS Different genetic components in the Ethiopian population, identified by mtDNA and Y-chromosome polymorphisms Am J Hum Genet 1998 62 420 434 9463310 10.1086/301702 Hasegawa M Di Rienzo A Kocher TD Wilson AC Toward a more accurate time scale for the human mitochondrial DNA tree J Mol Evol 1993 37 347 354 8308904 10.1007/BF00178865 Wakeley J Substitution rate variation among sites in hypervariable region 1 of human mitochondrial DNA J Mol Evol 1993 37 613 623 8114114 10.1007/BF00182747 Quintana-Murci L Chaix R Wells S Behar D Sayar H Scozzari R Rengo C Al-Zahery N Semino O Santachiara-Benerecetti A Where West meets East: The complex mtDNA landscape of the Southwest and Central Asian corridor Am J Hum Genet 2004 74 827 845 15077202 10.1086/383236 Kivisild T Rootsi S Metspalu M Metspalu E Parik J Kaldma K Usanga E Mastana S Papiha S Villems R Bellwood P, Renfrew C The Genetics of Language and Farming Spread in India Examining the farming/language dispersal hypothesis 2003 Cambridge: The McDonald Institute for Archaeological Research 215 222 Cordaux R Saha N Bentley G Aunger R Sirajuddin S Stoneking M Mitochondrial DNA analysis reveals diverse histories of tribal populations from India Eur J Hum Genet 2003 3 253 264 12678055 10.1038/sj.ejhg.5200949 Guha B The racial affinities of the people of India Census of India, 1931, Part III Ethnographical 1935 Simla: Government of India Press Richards M Rengo C Cruciani F Gratrix F Wilson J Scozzari R Macaulay V Torroni A Extensive female-mediated gene flow from sub-Saharan Africa into near eastern Arab populations Am J Hum Genet 2003 72 1058 1064 12629598 10.1086/374384 Tambets K Kivisild T Metspalu E Parik J Kaldma K Laos S Tolk H-V Gölge M Demirtas H Geberhiwot T Renfrew C, Boyle K The topology of the maternal lineages of the Anatolian and Trans-Caucasus populations and the peopling of the Europe: some preliminary considerations Archaeogenetics: DNA and the population prehistory of Europe 2000 Cambridge: Cambridge University Press 219 235 Underhill PA Passarino G Lin AA Shen P Mirazon Lahr M Foley R Oefner PJ Cavalli-Sforza LL The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations Ann Hum Genet 2001 65 43 62 11415522 10.1046/j.1469-1809.2001.6510043.x Reidla M Kivisild T Metspalu E Kaldma K Tambets K Tolk H Parik J Loogvali E Derenko M Malyarchuk B Origin and Diffusion of mtDNA Haplogroup X Am J Hum Genet 2003 73 1178 1190 14574647 10.1086/379380 Mountain JL Hebert JM Bhattacharyya S Underhill PA Ottolenghi C Gadgil M Cavalli-Sforza LL Demographic history of India and mtDNA-sequence diversity Am J Hum Genet 1995 56 979 992 7717409 Herrnstadt C Elson JL Fahy E Preston G Turnbull DM Anderson C Ghosh SS Olefsky JM Beal MF Davis RE Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups Am J Hum Genet 2002 70 1152 1171 11938495 10.1086/339933 Slatkin M Hudson RR Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations Genetics 1991 129 555 562 1743491 Majumder PP Ethnic populations of India as seen from an evolutionary perspective J Biosci 2001 26 533 545 11779963 Higham C Bellwood P, Renfrew C Languages and Farming Dispersals: Austroasiatic Languages and Rice Cultivation Examining the farming/language dispersal hypothesis 2003 Cambridge: The McDonald Institute for Archaeological Research Finnilä S Lehtonen MS Majamaa K Phylogenetic network for European mtDNA Am J Hum Genet 2001 68 1475 1484 11349229 10.1086/320591 Bermisheva M Tambets K Villems R Khusnutdinova E Diversity of mitochondrial DNA haplotypes in ethnic populations of the Volga-Ural region of Russia Mol Biol (Mosk) 2002 36 990 1001 12500536 Global land environments since the last interglacial: Comas D Plaza S Wells RS Yuldaseva N Lao O Calafell F Bertranpetit J Admixture, migrations, and dispersals in Central Asia: evidence from maternal DNA lineages Eur J Hum Genet 2004 12 495 504 14872198 10.1038/sj.ejhg.5201160 Papiha SS Chahal SM Mastana SS Variability of genetic markers in Himachal Pradesh, India: variation among the subpopulations Hum Biol 1996 68 629 654 8908795 Passarino G Semino O Pepe G Shrestha SL Modiano G Santachiara Benerecetti AS MtDNA polymorphisms among Tharus of eastern Terai (Nepal) Gene Geogr 1992 6 139 147 1339489 Papiha SS Genetic variation in India Hum Biol 1996 68 607 628 8908794 Singh KS ed The Scheduled Tribes 1997 Oxford: Oxford University Press Papiha SS Schanfield MS Chakraborty R Immunoglobulin allotypes and estimation of genetic admixture among populations of Kinnaur District, Himachal Pradesh, India Hum Biol 1996 68 777 794 8908801 Thangaraj K Ramana GV Singh L Y-chromosome and mitochondrial DNA polymorphisms in Indian populations Electrophoresis 1999 20 1743 1747 10435442 10.1002/(SICI)1522-2683(19990101)20:8<1743::AID-ELPS1743>3.0.CO;2-V Prasad B Ricker C Watkins W Dixon M Rao B Naidu J Jorde L Bamshad M Mitochondrial DNA variation in Nicobarese Islanders Hum Biol 2001 73 715 725 11758691 Thangaraj K Singh L Reddy A Rao V Sehgal S Underhill P Pierson M Frame I Hagelberg E Genetic affinities of the andaman islanders, a vanishing human population Current Biology 2003 13 86 93 12546781 10.1016/S0960-9822(02)01336-2 Melton T Clifford S Martinson J Batzer M Stoneking M Genetic evidence for the proto-Austronesian homeland in Asia: mtDNA and nuclear DNA variation in Taiwanese aboriginal tribes Am J Hum Genet 1998 63 1807 1823 9837834 10.1086/302131 Yao Y Nie L Harpending H Fu Y Yuan Z Zhang Y Genetic relationship of Chinese ethnic populations revealed by mtDNA sequence diversity Am J Phys Anthropol 2002 118 63 76 11953946 10.1002/ajpa.10052 Horai S Murayama K Hayasaka K Matsubayashi S Hattori Y Fucharoen G Harihara S Park KS Omoto K Pan IH mtDNA polymorphism in East Asian Populations, with special reference to the peopling of Japan Am J Hum Genet 1996 59 579 590 8751859 Oota H Settheetham-Ishida W Tiwawech D Ishida T Stoneking M Human mtDNA and Y-chromosome variation is correlated with matrilocal versus patrilocal residence Nat Genet 2001 29 20 21 11528385 10.1038/ng711 Fucharoen G Fucharoen S Horai S Mitochondrial DNA polymorphisms in Thailand J Hum Genet 2001 46 115 125 11310578 10.1007/s100380170098 Comas D Calafell F Mateu E Perez-Lezaun A Bosch E Martinez-Arias R Clarimon J Facchini F Fiori G Luiselli D Trading genes along the silk road: mtDNA sequences and the origin of Central Asian populations Am J Hum Genet 1998 63 1824 1838 9837835 10.1086/302133 Sambrook J Fritsch EF Maniatis T Molecular cloning: a laboratory manual., 1989 2 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press Kaessmann H Heissig F von Haeseler A Paabo S DNA sequence variation in a non-coding region of low recombination on the human X chromosome Nat Genet 1999 22 78 81 10319866 10.1038/8785 Bandelt H-J Forster P Sykes BC Richards MB Mitochondrial portraits of human populations using median networks Genetics 1995 141 743 753 8647407 Bandelt H-J Forster P Röhl A Median-joining networks for inferring intraspecific phylogenies Mol Biol Evol 1999 16 37 48 10331250 Free Phylogenetic Network Analysis Shareware Software: NETWORK, Forster P Harding R Torroni A Bandelt H-J Origin and evolution of Native American mtDNA variation: a reappraisal Am J Hum Genet 1996 59 935 945 8808611 Saillard J Forster P Lynnerup N Bandelt H-J Nørby S mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion Am J Hum Genet 2000 67 718 726 10924403 10.1086/303038 Rosenberg MS PASSAGE. Pattern Analysis, Spatial Statistics, and Geographic Exegesis. Version 1.1 Department of Biology, Arizona State University, Tempe, AZ 2001 Rosenberg M Passage software packet: Dupanloup I Bertorelle G Inferring admixture proportions from molecular data: extension to any number of parental populations Mol Biol Evol 2001 18 672 675 11264419

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-571531765610.1186/1471-2164-5-57Research ArticleGene expression variation between mouse inbred strains Turk Rolf [email protected]'t Hoen Peter AC [email protected] Ellen [email protected] Menezes Renée X [email protected] Meijer Emile J [email protected] Judith M [email protected] Ommen Gert-Jan B [email protected] Dunnen Johan T [email protected] Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, Nederland2 Department of Medical Statistics, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, Nederland2004 18 8 2004 5 57 57 13 5 2004 18 8 2004 Copyright © 2004 Turk et al; licensee BioMed Central Ltd.2004Turk et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays. Results Through ANOVA analysis with a false discovery rate of 10%, we show that 1.4% of the analysed genes is significantly differentially expressed between these mouse strains. Differential expression of several of these genes has been confirmed by quantitative RT-PCR. The number of genes affected by genetic background is approximately ten-fold lower than the number of differentially expressed genes caused by a dystrophic genetic defect. Conclusions We conclude that evaluation of the effect of background on gene expression profiles in the tissue under study is an effective and sensible approach when comparing expression patterns in animal models with heterogeneous genetic backgrounds. Genes affected by the genetic background can be excluded in subsequent analyses of the disease-related changes in expression profiles. This is often a more effective strategy than backcrossing and inbreeding to obtain isogenic backgrounds. ==== Body Background Due to their isogenicity, inbred mouse strains demonstrate low biological variability within each strain[1,2]. Genetic variation between inbred strains is considerable and has recently been characterized in detail using single nucleotide polymorphisms[3]. Differences in genetic background between strains affect the gene expression levels of a subset of genes, which probably explains phenotypic differences. Indeed, several reports have been published in which gene expression profiles have been used as QTLs in genetic mapping studies to identify complex traits [4-6]. From literature [7-9], it appears that the subset of genes for which expression is significantly affected by genetic background is small. However, this has never been related to the extent of gene expression changes observed due to disease-causing mutations. We are studying differential gene expression between affected and healthy muscle in a range of murine models for neuromuscular disorders with different genetic backgrounds (Turk et al., manuscript in preparation). We, therefore, determined gene expression levels in hindlimb muscles from five frequently used wildtype mouse inbred strains, and compared these to the differential gene expression levels in affected muscle tissue from a mouse model (mdx) for Duchenne muscular dystrophy with healthy muscle tissue. Both the number of differentially expressed genes between strains as well as the fold-change levels are lower when compared to the differences found in affected versus healthy muscle tissue. Results Gene expression levels in hindlimb muscle tissue from five different inbred strains (CBA, BALB, BL6, DBA, and BL10) were determined. Total RNA from two individuals per strain was isolated, reversed transcribed, and subsequently labelled according to a recently developed protocol (adapted from Xiang et al., 2002), which requires an input of only 1 μg total RNA. Labelled cDNA was hybridised to murine microarrays containing 7,776 65-mer oligonucleotides spotted in duplicate. Significance levels (p-values) between the five mouse inbred strains were calculated using analysis of variance[10]. Significance levels among two individual mice within each strain were determined using a hierarchical t-test providing higher statistical power than conservative methods for low (2–4) replicate numbers[11]. The higher power is yielded by borrowing information across genes to produce a better expression variance estimator. The gain in power is reported via an increase in the degrees of freedom associated with the t-test. Differentially expressed genes for both computations were selected by controlling the false discovery rate (FDR), as suggested by Benjamini and Hochberg (1995), rather than using pre-defined cut-offs for p-values or corrections for multiple testing. The FDR represents an expectation of the proportion of false positives among the selected differentially expressed genes, which increases dramatically during multiple testing, inherent in microarray experiments[12]. Using an FDR of 10% we selected 88 out of 6144 (1.4%) expressed genes that are differentially expressed between strains (Fig. 1). A lower number of differentially expressed genes was found in the analysis of variation within strains with identical FDRs of 10% (Table 1). Results with other FDR levels are available online as additional file. Correlation between gene expression levels of the two samples from each strain was high (Pearson correlation coefficient ranging from 0.87 to 0.95), also indicating low internal variation (Table 1). A considerable amount of differentially expressed genes (718 genes) were selected when pre-defined cut-off values (p < 0.05) were used to determine the differential gene expression between strains. However, adjusted FDR levels indicated a proportion of false positives equal to 42%. On the other hand, adjusting for multiple testing using Bonferroni correction proved to be too stringent, leaving no or few differentially expressed genes. Controlling the FDR, therefore, appears to be an optimal method for both selecting differential gene expression and simultaneously determining the validity of the experimental outcome. Figure 1 Differentially expressed genes between mouse inbred strains Relative expression levels of differentially expressed genes between mouse inbred strains are depicted in colour as relative intensity levels. Shown for each gene are GenBank accession number, description, functional annotation according to Gene Ontology, and UniGene cluster IDs. Relative expression levels are calculated by subtracting the average intensity value per gene from the strain-dependent intensity values. Differential expression was determined by selecting p-values from analysis of variance based on a false discovery rate of 10%. Table 1 Number of differentially expressed genes using several cut-off strategies Between strains Within strains MA-ANOVA Hierarchical t-test CBA BL10 BL6 DBA BALB Correlation 0.95 0.95 0.87 0.87 0.92 Naive (p < 0.05) 718 737 610 963 1043 483 Bonferroni 0 2 4 1 0 3 FDR 10% 88 2 4 14 0 16 Correlation between two individuals per strain was calculated using Pearson's correlation coefficient. Significance levels (p-values) between strains were calculated with MA-ANOVA, and within strains using the hierarchical t-test. Differential gene expression was determined by selecting genes with p-values lower than a specified threshold. Thresholds were selected using three different strategies; naive, Bonferroni corrected, and False Discovery Rate (10%), and resulted in different numbers of significantly differentially expressed genes. To put the influence of differential gene expression due to genetic background in perspective, we studied gene expression between affected and healthy tissue from hindlimb muscle derived from mdx mice, and from control mice with identical genetic backgrounds. Selection with an FDR of 10% resulted in 1298 differentially expressed genes. Differential gene expression between the two most divergent mouse inbred strains (BL6 and CBA, data not shown) was determined to allow a direct comparison with identical statistical methods. Selection with an FDR of 10% showed an approximately ten-fold decrease in the number of differentially expressed genes (126). Absolute fold changes were calculated and subsequently a comparison of the distribution was made (Fig. 2). Median gene expression levels are equal between affected/control and inbred/inbred. However, the number of large fold changes (>3) between affected/healthy (221) is much higher than between inbred/inbred (7), consistent with low contribution of differential expression due to genetic background. Figure 2 Effect of different genetic background on differential gene expression The distribution of absolute fold changes of differentially expressed genes (n = 1298) between affected (mdx) and healthy (WT) muscle were compared to the distribution of absolute fold changes of differentially expressed genes (n = 126) between two mouse inbred strains (CBA and BL6). Selections were based on a FDR of 10%. Although overall expression levels are similar between strains, a relatively high number of differentially expressed genes was due to deviating gene expression levels in BL6. We performed quantitative real-time RT-PCR (qPCR) on five genes to verify our microarray data. Two genes myomesin 1 and tropomodulin 1, which were 2.2-fold and 1.8-fold lower expressed in BL6 compared to the other strains on our microarrays, were also found to be lower expressed (2.0-fold and 2.2-fold respectively) in our qPCR assay (Fig. 3). Three other genes (dysferlin, cystatin B, and thrombospondin 4) showed no differential expression between any strains. Figure 3 Validation of BL6-dependent gene expression with qPCR Relative gene expression levels between mouse inbred strains of tropomodulin 1 (Tmod1) and myomesin 1 (Myom1) as determined by quantitative RT-PCR. Significantly lower expression (p < 0.01, marked by *) for both genes was shown in BL6 compared to other strains. Discussion This study shows that variation in overall gene expression levels between mouse inbred strains is relatively low in hindlimb muscle tissue. This is particularly evident when the number of differentially expressed genes between two mouse inbred strains (C57 vs. Bl6, 126 genes with 7 genes having a fold-change > 3) is compared to that between diseased and healthy muscle tissue (mdx vs. wild-type, 1298 genes with 221 genes having a fold-change >3). Therefore, the use of mice with deviating genetic background may be justified in disease-related studies. Alternatively, strain-dependent gene expression differences may be evaluated in the initial study phase of gene targeting experiments, although the effect of hybrid backgrounds is difficult to assess. Gene expression studies in the brain revealed that approximately 1% of expressed genes differ between two mouse strains[8]. Application of alternative statistical methods, similar to those used in our study, on this dataset resulted in an increase in the number of differentially expressed genes (approx. 3%) between the two mouse strains[7], demonstrating that the number of differentially expressed genes is highly dependent on the statistical criteria used. A similar number of differentially expressed genes was found in a comparison of hippocampal gene expression between 8 different mouse strains[9]. The results of our study in muscle tissue demonstrated that approximately 1.4% of the expressed genes show differential expression between mouse strains. Based on these results, strain differences in gene expression seem to have a similar magnitude across different tissues. Genomic variability could be correlated with high levels of single nucleotide polymorphisms (SNPs) occurring in specific blocks between mouse inbred strains. The presence of cis-acting (single nucleotide) polymorphisms may be associated with regulatory variation affecting gene expression levels. It was estimated that probably a consistent amount (up to 6%) of the roughly estimated 35,000 mouse genes contain such functional regulatory variants[13]. We investigated if differentially expressed genes were localized in blocks with high genomic variability, but our number of differentially expressed genes was too low to obtain statistically significant answers (data not shown). This study suggests an additional method for phenotyping mouse inbred strains and provides a list of genes with significant differential expression based upon false discovery rate selection. Although overall gene expression profiles are highly similar, most significant differences are determined by low gene expression levels of BL6 compared to the other strains. A large proportion of these BL6-specific genes function as structural muscle proteins (i.e. nebulin, alpha-actinin 2, myomesin 1 and radixin). To date, however, no major differences in muscle physiology in BL6-mice have been described which can be attributed to these reduced gene expression levels. Perfectly isogenic backgrounds are sometimes difficult to obtain. This explorative study demonstrates that the effect of genetic background on muscle expression profiles is significant but rather limited compared to other effects, e.g. the dystrophic genetic defect (mdx) we study. As such, the genetic background will only marginally interfere with data analysis. Determination of gene expression profiles between mouse strains enables flagging a modest number of differentially expressed genes, and is an efficient and sensible approach to circumvent tedious backcrossings, necessary to obtain isogenic animals. Methods Mouse breeding, tissue preparation and total RNA isolation We obtained CBA/CaOlaHsd (CBA), BALB/cOlaHsd (BALB), C57Bl/6JOlaHsd (BL6), DBA/2OlaHsd (DBA), and C57Bl/10ScSnOlaHsd (BL10) mice from Harland Laboratories, and C57Bl/10ScSn-Dmdmdx/J (mdx) mice from Jackson Laboratory at the age of 6 weeks. Mice were kept under standard conditions and were sacrificed by cervical dislocation when 8 weeks old. Hindlimb muscles (m. quadriceps femoris) were dissected and promptly snap-frozen in isopentane at -80°C. Total RNA was prepared by disrupting tissue using mortar and pestle and subsequent homogenisation by a rotor-stator homogenizor (Ultra-Turrax T25, Janke & Kunkel IKA-Labortechnik) in RNA-Bee (Campro Scientific) until uniformly homogenous (15–45 sec). Total RNA was isolated according to manufacturer's instructions followed by purification using RN-easy columns (Qiagen). Quality and yield was determined using Lab-on-a-chip (BioAnalyzer, Agilent). Target preparation and hybridisation Aminoallyl labelled cDNA (aa-cDNA) was prepared based on a previously described protocol[14]. Aliquots of 1 μg of total RNA in the presence of 2 μg amino-TN6 primer (5'-NH2-(CH2)6-TN6, Eurogentec) were adjusted to a volume of 21 μl with DEPC-treated H2O (diethyl pyrocarbonate, Sigma), heated for 10 minutes at 70°C and chilled on ice for 10 minutes. Reverse transcription mastermix (1.8 μl RevertAid RNaseH-M-MuLV reverse transcriptase (200 U/μl, MBI Fermentas), 6 μl 5x first-strand buffer (MBI Fermentas), and 1.2 μl 25x aa-dUTP / dNTP solution (2 μl 50 mM dATP, 2 μl 50 mM dCTP, 2 μl 50 mM dGTP, 1.2 μl 50 mM dTTP, 0.8 μl 50 mM aminoallyl-dUTP (Ambion)) was added per reaction and incubated at room temperature for 10 minutes followed by 2 hours at 42°C. RNA was hydrolysed by addition of 10 μl 0.5 M EDTA and 10 μl 1 M NaOH and incubation at 65°C for 30 minutes followed by neutralization by addition of 10 μl 1 M HCl. Aminoallyl labelled cDNA was then purified by combining 300 μl of PB-buffer (Qiagen) to 60 μl of the neutralized sample and centrifuged through a Qiaquick column (Qiagen) at 13000 rpm for 1 minute. Two washing steps were performed by spinning 500 μl of 75% EtOH at 13000 rpm for 1 minute while discarding the flow-through. To remove ethanol-traces the columns were centrifuged for an additional minute. cDNA was recovered by eluting three times using 30 μl basic H2O (3.3 mM NaHCO3 buffer, pH 9.0) and concentrated to a volume of 6.66 μl using a speedvac. Aliquots of Cy3 and Cy5 reactive dyes (PA23001, PA25001, Amersham) were prepared by dissolving each vial of monoreactive dye in 40 μl fresh anhydrous DMSO (Sigma) and dividing into aliquots of 2 μl followed by vaccuumdrying until dry and subsequent storage at 4°C in the presence of silica. Fluorescent dyes were coupled by adding 3.33 μl of bicarbonate buffer (1 M NaHCO3 buffer, pH 9.0) to the aa-cDNA sample and dissolving the dried aliquot of reactive dye, followed by incubation at room temperature for 1 hour in the dark. To the samples 4.5 μl 4 M hydroxylamine (Sigma) was added and incubated at room temperature in the dark for 15 minutes, followed by addition of 186 μl TE-3-buffer. Hybridisation mixtures were prepared by combining a Cy3-labeled cDNA sample with a Cy5-labeled cDNA sample and 10 μl Mouse-Hybloc (1 μg/μl, Applied Genetics Laboratories) followed by removing uncoupled dyes by spinning through a pre-wetted Microcon column (YM30, Amicon) for 8 minutes at 13000 rpm. Hybridisation mixture was washed by spinning 500 μl TE-3-buffer through the column and discarding the flow-through. This step was repeated two times as 2 μl yeast-tRNA (10 μg/μl, Sigma) and 2 μl polyA-RNA (10 μg/μl, Sigma) were added during the last step. Mixture was collected by inverting the column and spinning for 1 minute at 13000 rpm. Hybridisation mixture was finalized by adding TE-3-buffer to 84 μl together with 17 μl 20x SSC and 3 μl 10% SDS followed by denaturing at 100°C for 2 minutes, renaturing at room temperature for 15 minutes and spinning at 13000 rpm for 10 minutes. Labelled target was hybridised overnight on murine oligonucleotide microarrays (65-mer with 5'-hexylaminolinker, Sigma-Genosys mouse 7.5 K oligonucleotide library, spotted in duplicate). Hybridisation occurred in a automatic hybridisation station (GeneTac, Perkin Elmer) and was followed by washing with 5x 2xSSC + 0.1% SDS at 30°C, 5x 1xSSC at 30°C, 3x 0.2xSSC at 30°C, 1x 0.2xSSC at 65°C, 2x 0.2xSSC at 30°C, and subsequently scanned as described previously[15]. Experimental design, data extraction and analysis Gene expression profiles from hindlimb muscle derived from 2 male animals of each strain were generated using dye-swap experiments. Subsequent duplicate spots on each array resulted in 8 replicate measurements per gene. Targets were assigned at random to the arrays, while avoiding co-hybridisation of samples from the same strain. GenePix Pro 3.0 (Axon) was used for feature extraction and quantification. Genes were considered as being expressed when the corresponding feature was not flagged by the algorithm provided by GenePix. Local background corrected spot intensities were normalized using Variance Stabilization and Normalization (VSN) in R [16]. Array data has been made available through the GEO data repository of the National Center for Biotechnology Information under series GSE662. Correlation between individuals was calculated using Pearson's correlation coefficient. Significantly differential expression levels were determined using MA-ANOVA (MAANOVA2.0 The Jackson Laboratory ), hierarchical t-test [11] and the False Discovery Rate [17] selection procedure. Quantitative Reverse Transcription Polymerase Chain Reaction qPCR was performed in duplicate for each individual resulting in four measurements per strain per gene. cDNA was prepared by reverse transcription using 1 μg total RNA as template. Random hexamers (40 ng) were used to prime the transcription after heating 10 minutes at 70°C followed by chilling on ice for 10 minutes. cDNA was synthesized by RevertAid RNaseH- MuLV reverse transcriptase and accompanying buffer (MBI-Fermentas) using 1 mM dNTPs. The mixture was incubated at room temperature for 10 minutes before a 2 hour incubation step at 42°C, followed by 10 minutes at 70°C. Quantitative PCR was performed using the Lightcycler (Roche). PCR mixture was prepared by combining cDNA dilution, 10 pmol forward and reverse primer, MgCl2 (4 mM) with 4x home-made LC mastermix (0.9 mM dNTPs, BSA (1 μl/μl, Pharmacia Biotech), Taq polymerase (0.8 U/μl), 4x SYBR Green I (Molecular Probes), 4x AmpliTaq Reaction Buffer (Perkin Elmer)) to a total volume of 20 μl. Amplicons were generated during 45 cycles with annealing temperature set at 55°C. Optimal cDNA dilutions and relative concentrations were determined using a dilution series per gene. Replicate experiments (n = 4) were normalized to 1 and relative expression values were determined by calculating the ratio per gene over the average relative expression of genes, which show no differential expression on both microarray and qPCR (dysferlin, cystatin B, and thrombospondin 4). Significance levels were calculated with a one-sample t-test. PCR primer pairs were designed using the Primer3 search engine, available at: Primer3 Software Distribution . The screened genes and the oligonucleotide primer pairs used for each of the genes in this study correspond to the following nucleotides: myomesin1, 4761–4780 and 4865–4884 (NM_010867); tropomodulin1, 670–689 and 878–897 (NM_021883); dysferlin, 4218–4237 and 4353–4372 (AF188290); cystatinB, 3–22 and 151–170 (NM_007793); thrombospondin4, 2167–2186 and 2289–2308 (NM_011582). Authors' contributions RT carried out the tissue preparation, total RNA isolation, target preparation, hybridisations, experimental design, data extraction, data analysis, rt-PCR, and the drafting of the manuscript. PH participated in the experimental design, analysis, rt-PCR, and study coordination. ES participated in the experimental design and analysis. RM provided statistical support. EM was responsible for mouse breeding and tissue preparation. JM participated in experimental design. GO and JD coordinated the study. All authors read the final manuscript. Supplementary Material Additional File 1 Differentially expressed genes between mouse inbred strains are selected with a false discovery rate of 10, 15, and 20%. Selected genes are indicated with 1, genes not selected by the specified criteria are indicated with 0. The mean of the relative gene expression levels of each of the five mouse strains is shown. For each gene the GenBank accession number is shown as well as the UniGene ID, gene description and the gene ontology description. The additional file is formatted as Comma Separated Values (CSV) file, and is named Turketal2004_Additional_File.csv. Click here for file Acknowledgements We thank Claire Wade and Mark Daly (Whitehead/MIT Center for Genome Research) for providing SNP density levels, and Stefan White (LUMC) for critical comments on the manuscript. This work was supported by the Nederlandse Stichting voor Wetenschappelijk Onderzoek (NWO), the Center for Medical Systems Biology (CMSB) established by the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research (NGI/NWO), and the Muscular Dystrophy Campaign (UK). Technical support was provided by the Leiden Genome Technology Center. ==== Refs Beck JA Lloyd S Hafezparast M Lennon-Pierce M Eppig JT Festing MF Fisher EM Genealogies of mouse inbred strains Nat Genet 2000 24 23 25 10615122 10.1038/71641 Pritchard CC Hsu L Delrow J Nelson PS Project normal: defining normal variance in mouse gene expression Proc Natl Acad Sci U S A 2001 98 13266 13271 11698685 10.1073/pnas.221465998 Wade CM Kulbokas EJ,III Kirby AW Zody MC Mullikin JC Lander ES Lindblad-Toh K Daly MJ The mosaic structure of variation in the laboratory mouse genome Nature 2002 420 574 578 12466852 10.1038/nature01252 Eaves IA Wicker LS Ghandour G Lyons PA Peterson LB Todd JA Glynne RJ Combining mouse congenic strains and microarray gene expression analyses to study a complex trait: the NOD model of type 1 diabetes Genome Res 2002 12 232 243 11827943 10.1101/gr.214102. Article published online before print in January 2002 Schadt EE Monks SA Drake TA Lusis AJ Che N Colinayo V Ruff TG Milligan SB Lamb JR Cavet G Linsley PS Mao M Stoughton RB Friend SH Genetics of gene expression surveyed in maize, mouse and man Nature 2003 422 297 302 12646919 10.1038/nature01434 Chesler EJ Wang J Lu L Qu Y Manly KF Williams RW Genetic correlates of gene expression in recombinant inbred strains: a relational model system to explore neurobehavioral phenotypes Neuroinformatics 2003 1 343 357 15043220 10.1385/NI:1:4:343 Pavlidis P Noble WS Analysis of strain and regional variation in gene expression in mouse brain Genome Biol 2001 2 RESEARCH0042 11597334 10.1186/gb-2001-2-10-research0042 Sandberg R Yasuda R Pankratz DG Carter TA Del Rio JA Wodicka L Mayford M Lockhart DJ Barlow C Regional and strain-specific gene expression mapping in the adult mouse brain Proc Natl Acad Sci U S A 2000 97 11038 11043 11005875 10.1073/pnas.97.20.11038 Fernandes C Paya-Cano JL Sluyter F D'Souza U Plomin R Schalkwyk LC Hippocampal gene expression profiling across eight mouse inbred strains: towards understanding the molecular basis for behaviour Eur J Neurosci 2004 19 2576 2582 15128411 10.1111/j.0953-816X.2004.03358.x Kerr MK Martin M Churchill GA Analysis of variance for gene expression microarray data J Comput Biol 2000 7 819 837 11382364 10.1089/10665270050514954 de Menezes RX Boer JM Houwelingen JC Microarray data analysis: a hierarchical t test to handle heteroscedasticity Applied Bioinformatics 2004 in press Reiner A Yekutieli D Benjamini Y Identifying differentially expressed genes using false discovery rate controlling procedures Bioinformatics 2003 19 368 375 12584122 10.1093/bioinformatics/btf877 Cowles CR Joel NH Altshuler D Lander ES Detection of regulatory variation in mouse genes Nat Genet 2002 32 432 437 12410233 10.1038/ng992 Xiang CC Kozhich OA Chen M Inman JM Phan QN Chen Y Brownstein MJ Amine-modified random primers to label probes for DNA microarrays Nat Biotechnol 2002 20 738 742 12089562 10.1038/nb0702-738 't Hoen PA de Kort F van Ommen GJ den Dunnen JT Fluorescent labelling of cRNA for microarray applications Nucleic Acids Res 2003 31 e20 12595569 10.1093/nar/gng020 Huber W Von Heydebreck A Sultmann H Poustka A Vingron M Variance stabilization applied to microarray data calibration and to the quantification of differential expression Bioinformatics 2002 18 S96 S104 12169536 Benjamini Y Hochberg Y Controlling the false discovery rate: a practical and powerful appraoch to multiple testing. J Roy Stat Soc B 1995 57 289 300

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BMC Genomics. 2004 Aug 18; 5:57

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BMC Genomics

10.1186/1471-2164-5-57

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-601533314110.1186/1471-2164-5-60SoftwarePOSA: Perl Objects for DNA Sequencing Data Analysis Aerts Jan A [email protected] Bart J [email protected] Martien AM [email protected] Animal Breeding and Genetics Group, Wageningen University, PO Box 338, 6700AH Wageningen, Netherlands2 Complex Genetics Group, Department of Biomedical Genetics, University Medical Centre, PO Box 80030, 3508 TA Utrecht, Netherlands2004 27 8 2004 5 60 60 23 6 2004 27 8 2004 Copyright © 2004 Aerts et al; licensee BioMed Central Ltd.2004Aerts et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Capillary DNA sequencing machines allow the generation of vast amounts of data with little hands-on time. With this expansion of data generation, there is a growing need for automated data processing. Most available software solutions, however, still require user intervention or provide modules that need advanced informatics skills to allow implementation in pipelines. Results Here we present POSA, a pair of new perl objects that describe DNA sequence traces and Phrap contig assemblies in detail. Methods included in POSA include basecalling with quality scores (by Phred), contig assembly (by Phrap), generation of primer3 input and automated SNP annotation (by PolyPhred). Although easily implemented by users with only limited programming experience, these objects considerabily reduce hands-on analysis time compared to using the Staden package for extracting sequence information from raw sequencing files and for SNP discovery. Conclusions The POSA objects allow a flexible and easy design, implementation and usage of perl-based pipelines to handle and analyze DNA sequencing data, while requiring only minor programming skills. ==== Body Background Today, many genetics laboratories have access to modern capillary DNA sequencing machines, such as the ABI PRISM 3100, 3700 or 3730. These machines generate vast amounts of raw sequence data with little user intervention. Consequently, the amount of data to be analyzed has expanded and the bottleneck now is the analysis capacity. Data analysis capacity can be increased by higher levels of automation. Investments in infrastructures to process the raw sequencing data in sophisticated but rigid pipelines might be justified for larger laboratories and larger projects but might be too costly for smaller laboratories. In addition, rigid pipelines are too impractical if different projects share run-time on the same machine while requiring (slightly) different analysis procedures (e.g. vector trimming is needed in plasmid sequencing, but needless when sequencing PCR products). Nucleotide sequence analysis can be performed with a variety of software tools. Although the number of console and web-based software tools has grown rapidly, the routine use of data input, output and storage may be inconvenient. Furthermore, for performing a series of analyses with different software tools, the sequence data need to be reformatted to the required data structure. Alternatively, sophisticated software suites that provide an integrated environment often are expensive. Several of the available software solutions are designed to facilitate automated DNA sequence analysis at low cost. Well-known solutions are the Staden package and Bioperl. The Staden Package contains pregap4 and gap4, full-featured applications with an intuitive graphical user interface [1]. These programs handle a list of raw sequence reads method-by-method. The programs in the Staden Package typically require a degree of user intervention and thus hands-on time. Alternatively, Bioperl is a group of perl modules describing many genetics and genomics concepts [2]. For example, it includes the Bio::Seq::SeqWithQuality object that provides some of the basic properties of a raw sequence (i.e. its nucleotide sequence and quality values); the Bio::Tools::Primer3 object provides methods to work with primer3 input and output. However, to build custom DNA sequencing data pipelines, basic programming skills are needed to combine all these modules. Smaller laboratory sites, however, often need to implement versatile pipelines that can be adjusted for any research question that suits the project best; at the same time, they often also do not have dedicated programmers available. Although (semi-)automated procedures have been published by other groups [3,4], these are mostly focused on one particular pipeline and environment. Here, we present POSA, a set of two new perl objects (Read.pm and Contig.pm) that describe a raw sequence and a Phrap contig in detail and are easily implemented in perl-based pipelines. Because these objects provide building blocks for sequencing data analysis pipelines and the actual pipelines are built using perl-scripts, the POSA objects can be used in very diverse settings. Implementation The POSA source code is entirely coded in object-oriented Perl and consists of two objects: Read.pm and Contig.pm. In general, there are two important concepts associated with objects: methods (built-in procedures that can be performed on the object) and properties (describing some of the characteristics of the object). Most methods in the objects rely on the availability of other third-party programs (see Dependencies). Basically, POSA provides a wrapper around these programs and provides easy design and implementation of these programs in automated data analysis. The Read.pm object describes a DNA sequence trace and includes methods for data import from a variety of formats. It relies on Phred [5,6] for import and interpretation of raw sequence data. The original trace data are stored in binary (scf) format within the object. Other methods of Read.pm use modules of the Staden Package [1], such as qclip and vector_clip (if installed). Properties of Read.pm include e.g. the DNA sequence, quality scores, template and vector names and read direction. The Contig.pm object contains a method to assemble contigs of reads using the Phrap program [6]. The object typically is created based on a list of Read.pm objects and can be exported as alignments or screened for polymorphisms using PolyPhred [7]. Both the Read.pm and Contig.pm objects were designed with flexibility in mind. To allow a (virtually) unlimited amount of data to be processed, the perl scripts using these objects work sequence-by-sequence rather than method-by-method. Typically, these objects are called from straightforward perl scripts that outline the analysis steps to be performed. Example scripts using the objects can be accessed from the download website. An example of a script and output using the two objects to process a set of reads and annotate sequence polymorphisms from the assembled contig is given in Figure 1 and Figure 2. Figure 1 A typical script that takes a list of ab1 files for analysis and assembly, reports the contig, and lists the putative SNP positions and SBE primers. Figure 2 Typical output as generated by the script in Figure 1. POSA was developed with perl 5.6.1 and tested on a SuSE linux 8.1 system for abi-files from the ABI PRISM 377 DNA Sequencer and 3100 Genetic Analyzer (Applied Biosystems). Phred, Phrap and PolyPhred versions were 0.000925.c, 0.990329 and 4.05, respectively. Results and Discussion Functionality POSA provides an interface to design and implement automated sequencing data analysis. Sequencing data may be used in a variety of formats and originate from a variety of sources, e.g. data in fasta, abi/ab1 or scf format retrieved from websites or from newly generated traces. In addition, new objects can be initiated from a text file or can be opened from previous stored objects. Subsequently, a variety of methods can be applied, including basecalling and assessment of quality codes (by Phred), quality clipping, vector clipping, screening for E. coli (or other) sequence, contig assembly (by Phrap) and analysis. The method asPrimer3 can automatically generate input for the primer3 program [8] and is available in both objects. To facilitate automated SNP discovery or typing, the SearchSnps method will generate output as shown in Figure 2. This method is based on the PolyPhred program and uses the 'rank' argument to set the stringency. Finally, data can be stored in objects, or in files in either exp, scf or fasta format. In addition, the data can be saved in a primer3 input file to allow automated PCR primer design, or data can be saved in MIPE format (i.e. an XML format to store information on PCR experiments; see ). Data on assembled contigs can be exported as a list of reads in a contig, as consensus sequence, as alignment, as putative SNPs, as SBE primers for SNP genotyping or as gff file for visualization in Gbrowse [9]. Combinations of the diversity of input, analysis and output options allow for a wide spectrum of possible implementations. Examples of possible analysis pipelines include (but are not limited to) BAC-end sequencing with automated PCR primer design for chromosome walking and resequencing of PCR products with SNP annotation either for SNP genotyping or for SNP discovery and SBE primer design. Examples of scripts are provided on the web site . Performance Although it represents only one of the numerous possible POSA-based pipelines, performance of POSA was validated by comparison of SNP discovery with the data after analysis using the Staden package. To do so, 5 PCR products were resequenced from a panel of 16 individuals to identify SNPs. Manual editing using the Staden Package revealed a total of 48 SNPs. Automated analysis using POSA also yielded a total of 48 SNPs with SNP ranking codes 1-3. Together, 41 SNPs were assigned with both manual editing and POSA. The remaining 7 SNPs assigned in manual editing corresponded to SNPs with ranks 4-6 in the POSA analysis. The 7 SNPs that were only assigned by POSA all originated from regions with lower quality sequence. While analysis time was reduced from several hours to a few minutes, POSA assigned SNPs in a way that was highly consistent with manual editing. This was expected because POSA provides options for an integrated analysis pipeline, but essentially is a wrapper around well-established sequence analysis tools like Phred, Phrap and PolyPhred. Intended use and benefits for users POSA is a tool that provides easy and highly automated DNA sequence and contig data analysis using popular analysis tools. Automated sequence analysis reduces analysis time from several hours to a few minutes. Pipelines can easily be expanded or adapted through perl scripts. Writing or altering the perl scripts is straightforward to do for people with only basic computer skills, although more linux/unix experience might be necessary to install the required software (e.g. Phred and Phrap). Overall, this guaranties easy implementation of highly automated quality pipelines in combination with high flexibility in setup and design. The perl objects are released under an open source license, allowing code improvements by the user community. Conclusions POSA describes a DNA sequence read and a Phrap contig assembly in detail. These objects allow a flexible and easy setup of perl-based pipelines to handle DNA sequencing data, including generating primer3 input and automated SNP discovery, while requiring only little programming skills. Availability and requirements Project name: POSA Project home page: Operating system: platform independent Programming language: Perl 5.6.1 License: Artistic License (Open Source) Requirements • Perl modules: Carp; Statistics::Descriptive; Tie::File; IO::File; POSIX:: Storable. • Phred, Phrap, PolyPhred • Pregap4, gap4 (Staden Package (optional)) • Primer3 (optional) List of abbreviations POSA Perl objects for DNA sequencing data analysis SNP single nucleotide polymorphism abi/ab1ABI PRISM trace file format scf standard chromatogram format exp experiment file format, developed by Staden (see ) MIPE minimum information on PCR experiments (see ) BAC bacterial artificial chromosome PCR polymerase chain reaction SBE single base extension Authors' contributions JA programmed the Perl objects and participated in development of concept and architecture of the software; BJ participated in development of concept and architecture and wrote the manuscript; MG supervised the project. All authors read and approved the final manuscript. Acknowledgements The authors wish to thank Tineke Veenendaal for testing. ==== Refs Staden R Beal KF Bonfield JK The Staden package, 1998 Methods Mol Biol 2000 132 115 30 10547834 Stajich JE Block D Boulez K Brenner SE Chervitz SA Dagdigian C Fuellen G Gilbert JG Korf I Lapp H Lehvaslaiho H Matsalla C Mungall CJ Osborne BI Pocock MR Schattner P Senger M Stein LD Stupka E Wilkinson MD Birney E The Bioperl toolkit: Perl modules for the life sciences Genome Res 2002 12 1611 8 12368254 10.1101/gr.361602 Mao C Cushman JC May GD Weller JW ESTAP – an automated system for the analysis of EST data Bioinformatics 2003 19 1720 2 15593407 10.1093/bioinformatics/btg205 Xu H He L Zhu Y Huang W Fang L Tao L Zhu Y Cai L Xu H Zhang L Yu H Zhou Y EST Pipeline System: detailed and automated EST data processing and mining Geno Prot Bioinfo 1 236 242 Ewing B Hillier L Wendl MC Green P Base-calling of automated sequencer traces using phred. I. Accuracy assessment Genome Res 1998 8 175 85 9521921 Ewing B Green P Base-calling of automated sequencer traces using phred. II. Error probabilities Genome Res 1998 8 186 94 9521922 Nickerson DA Tobe VO Taylor SL PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing Nucleic Acids Res 1997 25 2745 51 9207020 10.1093/nar/25.14.2745 Rozen S Skaletsky H Primer3 on the WWW for general users and for biologist programmers Methods Mol Biol 2000 132 365 86 10547847 Stein LD Mungall C Shu SQ Caudy M Mangone M Day A Nickerson E Stajich JE Harris TW Arva A Lewis S The Generic Genome Browser: a building block for a model organism system database Genome Res 2002 12 1599 1610 12368253 10.1101/gr.403602

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BMC Genomics. 2004 Aug 27; 5:60

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BMC Genomics

10.1186/1471-2164-5-60

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==== Front BMC MicrobiolBMC Microbiology1471-2180BioMed Central London 1471-2180-4-341533934210.1186/1471-2180-4-34Research ArticleDam inactivation in Neisseria meningitidis: prevalence among diverse hyperinvasive lineages Jolley Keith A [email protected] Li [email protected] E Richard [email protected] Martin CJ [email protected] The Peter Medawar Building for Pathogen Research and Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3SY, UK2 Molecular Infectious Diseases Group, University of Oxford Department of Paediatrics, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK2004 31 8 2004 4 34 34 22 7 2004 31 8 2004 Copyright © 2004 Jolley et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background DNA adenine methyltransferase (Dam) activity is absent in many, but not all, disease isolates of Neisseria meningitidis, as a consequence of the insertion of a restriction endonuclease-encoding gene, the 'dam replacing gene' (drg) at the dam locus. Here, we report the results of a survey to assess the prevalence of drg in a globally representative panel of disease-associated meningococci. Results Of the known meningococcal hyper-invasive lineages investigated, drg was absent in all representatives of the ST-8 and ST-11 clonal complexes tested, but uniformly present in the representatives of the other hyper-invasive lineages present in the isolate collection (the ST-1, ST-4, ST-5, ST-32 and ST-41/44 clonal complexes). The patterns of sequence diversity observed in drg were consistent with acquisition of this gene from a source organism with a different G+C content, at some time prior to the emergence of present-day meningococcal clonal complexes, followed by spread through the meningococcal population by horizontal genetic exchange. During this spread a number of alleles have arisen by mutation and intragenic recombination. Conclusion These findings are consistent with the idea that possession of the drg gene may contribute to the divergence observed among meningococcal clonal complexes, but does not have a direct mechanistic involvement in virulence. ==== Body Background Neisseria meningitidis, the causative agent of meningococcal meningitis and septicaemia, is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population [1,2]. There is evidence for extensive horizontal genetic exchange in populations of this antigenically and genetically variable Gram-negative bacterium [3-7] but, despite the diversity of carried meningococci, only a limited number of genotypes – the hyper-invasive lineages – are responsible for most reported disease [8]. These lineages have been identified by the techniques of multilocus enzyme electrophoresis [9] and multilocus sequence typing (MLST) [10] as clonal complexes. In recent years, the ST-1 complex (formerly subgroup I), ST-4 complex (subgroup IV), and ST-5 complex (subgroup III) have dominated meningococcal disease in Africa and Asia, while members of the ST-11 (ET-37) complex, ST-8 complex (cluster A4), ST-41/44 complex (lineage 3), and ST-32 (ET-5) complex have caused most disease in other parts of the world. Meningococci occasionally cause epidemic outbreaks of varying scale up to global pandemics, although at a given point in time disease in a given geographical locale is often dominated by a limited number of clonal complexes [8]. DNA adenine methyltransferase (Dam) is an enzyme involved in the mismatch repair system of bacteria. During DNA replication, only the parental strand is fully methylated by the enzyme, since methylation is not immediate, allowing a mismatch on the newly synthesised strand to be excised and replaced [11]. Disruption of Dam activity suggests that mismatch repair will be less effective, potentially allowing the creation of frameshift mutations in the homopolymeric or simple repeat motifs within the promoter or coding regions of surface antigen genes leading to the reversible switching of their expression state. This is an important mechanism found at bacterial loci that encode gene products that are advantageous under certain conditions but not others; such genes have been termed contingency loci [12]. There is extensive evidence for such loci in the meningococcal genome [13]. In particular, the ability to vary the expression state of surface-exposed cellular components is important for within host adaptation, for example by allowing evasion of the immune response in the nasopharynx. These mechanisms are also important in the occasional transition from harmless colonisation to invasive disease [14-16]. Examples of such loci described in the meningococcus to date include: the capsular polysaccharide [17]; the Opc outer membrane protein [18-20]; pili [21]; the PorA protein [22]; and opacity proteins [19,23]. Dam methylation has been implicated in modifying the virulence of a number of bacterial pathogens [24-26], but its role in N. meningitidis has been contentious. Bucci et al., 1999 [27] suggested that the absence of Dam activity was responsible for high rates of phase variation in the siaD capsule gene, resulting in an increased virulence of some strains. In addition, the latter work suggested that all pathogenic isolates lacked Dam activity, the dam gene inactivated by the insertion of a putative restriction endonuclease, named 'dam replacing gene' (drg), with the genotypes dam+/drg- and dam-/drg+ being mutually exclusive. However, two subsequent studies [28,29] have found no effect. The drg gene encodes a restriction enzyme, NmeBII that is similar to the Streptococcus pneumoniae DpnI restriction endonuclease which cleaves at GmeATC, but not at GATC, sequences [30,31]. In the present study the prevalence of drg and its association with disease-associated isolates of N. meningitidis was examined by a survey of its distribution in a collection of isolates chosen to represent the known diversity of pathogenic meningococci [10]. The results suggest that, while drg is present in some lineages but not others, this gene has spread in the meningococcal population by horizontal genetic exchange, possibly after the introduction of the drg gene from an exogenous source. Results Distribution of dam and drg among invasive meningococci In all cases, there was an exact correlation with the presence of dam and the absence of drg, and vice-versa. Of the 84 isolates tested, 23 were indicated to be dam+/drg- and 61 to be dam-/drg+ by restriction analysis and PCR amplification (see Additional file 1). The presence of dam or drg was associated with particular clonal complexes. Of the hyper-invasive lineages included in the analysis, all ST-8 complex and ST-11 complex isolates were dam+/drg-, while all of the representatives of the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes were dam-/drg+. Diversity of drg gene fragment sequences The level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within drg gene fragments was similar to that seen with the housekeeping genes used for MLST (Table 1), with a total of 19 different fragment sequences (drg-1 – drg-19) present in this collection. The ratio of non-synonymous to synonymous substitutions (dN/dS, 0.11) was also similar to that seen in the MLST gene fragments. Three drg gene fragments (drg-13, drg-14 and drg-15) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons (Figure 1) and one fragment (drg-16) had an insertion corresponding to a 24-base direct repeat. The G+C content of drg at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1–57.4%. Split decomposition analysis of the drg fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the drg-2 allele (Figure 2). The distribution of the different drg sequences amongst the clonal complexes is also shown in Figure 2. Discussion The meningococcus is an example of an accidental pathogen, a commensal organism that rarely causes disease and which gains no evolutionary benefit from this process [32]. A complete understanding of the diseases caused by such organisms is dependent upon an appreciation of the mechanisms by which harmless carriage develops into invasive disease [33]. The observation that some meningococcal genotypes, the hyper-invasive lineages, are more likely to cause disease than others [8,10] suggests that comparative studies of disease-associated and carried meningococci may identify genetic factors responsible for disease [34]. In this context, the suggestion that the possession of a single genetic change, the insertion of the drg gene at the dam locus, was essential for virulence in meningococci [27] was attractive as it provided a single characteristic associated with the disease phenotype. Further, this proposal provided a plausible mechanistic explanation, namely the promotion of rapid switching of expression of contingency genes, for example those encoding the capsule which represents the best defined meningococcal virulence determinant [35]. This is consistent with the evidence that inactivation of Dam has been shown to affect rates of recombination and phase change in other species [25,36]. The data presented here, however, do not support the contention that the drg insertion is preferentially associated with disease-associated meningococcal isolates, even those expressing serogroup B capsular polysaccharide. There was no evidence of a difference between disease-associated and carried meningococcal isolates with 76% (45/59) of disease-associated isolates and 75% (11/15) of carrier isolates possessing the drg insertion. The isolate collection employed by the present investigation was chosen to be globally representative of meningococcal disease in the latter part of the 20th century, with multiple examples of each of the major hyper-invasive meningococci identified over this period. When analysed by clonal complex, which identifies hyper-invasive lineages, it was apparent that the drg insertion was absent from all of the isolates representing the ST-8 and ST-11 clonal complexes, both major hyper-invasive lineages but, conversely, drg was present in all representatives of the hyper invasive lineages represented by the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes. This association was independent of the serogroup expressed by the isolates and provided a likely explanation of the earlier observations [27]. As meningococcal disease in a given locale at a given time tends to be dominated by a limited number of clonal complexes [8], it is likely from the results provided here that a given sample of disease-associated isolates collected from a given locale will be uniform at the dam locus. At a given point in time they may be dominated by drg containing meningococci, while at other times isolates without this insertion might dominate. For example, as both the ST-1 and ST 32 complexes contain the drg insertion, this insertion would be prevalent among the disease isolates recovered by the pandemic outbreaks caused by these clonal complexes [37,38]; conversely, the spread of ST-11 meningococci [39,40] would result in an increased prevalence of disease-associated meningococci without the drg insertion. This observation highlights the importance of assembling genetically defined isolate collections with a prescribed sampling frame if comparisons of meningococci with potential for virulence are to be undertaken. It has been suggested that the drg gene has been introduced into Neisseria from an exogenous source [30]. The difference in the G+C% content of the drg gene compared to the meningococcal housekeeping genes was consistent with this idea; however, the levels of diversity among the drg alleles, which was similar to that observed in the meningococcal housekeeping genes, along with the reported occurrence of drg in other Neisseria species [30], suggested that this was a relatively old event predating the emergence of present-day clonal complexes. The ratio of non-synonymous to synonymous substitutions (dN/dS), which was also similar to that observed in meningococcal housekeeping genes, suggested that the drg gene had been subject to stabilising selection for conservation of function since its putative introduction into the meningococcal population, although the possession of frame shift mutations in some of the drg alleles demonstrated that, as with N. lactamica, not all meningococci possessing the drg insertion expressed the endonuclease [30]. In other respects the distribution of the gene among clonal complexes and the patterns of nucleotide sequence variation observed were similar to those seen in meningococcal housekeeping genes, suggesting that it is subject to similar selection pressures [41]. The presence of multiple methylation systems is a characteristic of the Neisseria and may be related to the transformable nature of these organisms [42]. Whole genome comparisons of meningococci belonging to different clonal complexes frequently yield different restriction modification systems as the principal detectable genetic differences [43,44]. It is attractive to speculate that the genetic isolation of clonal complexes may be promoted by such differences [45] and in this respect the insertion of drg in the dam locus, replacing a methylase with an endonuclease, may provide a potent barrier to genetic exchange from dam+ to drg+ meningococci; indeed, while there was evidence for the occasional horizontal genetic exchange of drg alleles among the genetically diverse meningococci that possess the insertion, there was no evidence for the transformational loss of drg from a given clonal complex among the isolates examined here. This concept of genetic isolation among lineages receives some support from the possession of porB2 alleles by members of the dam+ ST-8 and ST-11 complexes and porB3 alleles by the clonal complexes with the drg insertion [9,39], but the evidence for limitations in gene flow between these subgroups of meningococci is not at present conclusive. Conclusions While it is possible that the possession of the drg insertion may influence meningococcal population structure, the data presented here do not support a direct association of this genotype with meningococcal virulence. Methods Bacterial isolates The 84 isolates used in this study (see Additional file 1) were a subset of a collection of 107 diverse meningococci assembled to develop and validate the MLST scheme for Neisseria meningitidis [10]. The collection included representatives of the major hyper-invasive lineages and has been characterised at many genetic loci (full details of this isolate collection are available at ). Restriction digestion of chromosomal DNA The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases DpnI, DpnII, and Sau3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not: DpnI cleaves this target sequence only when N6-methyladenine is present within the recognition sequence; DpnII cleaves only unmethylated sites, and Sau3AI cleaves both methylated and unmethylated target sequences. PCR amplification and nucleotide sequence determination The presence of the dam gene was determined by polymerase chain reaction (PCR) performed using dam specific primers, DamF1 (5' – TAAAATGGGCAGGCGGCA – 3') and DamB2 (5' – CGTAAGGGGGATCGCAAT – 3'). These amplify a 534 bp fragment from the 5' end of the dam gene in dam+ strains but not from dam-strains. The presence of drg was determined by PCR using primers DrgF1 (5' – CATGAATTTATTTTTCGATA – 3') and DrgB2 (5' – AATTTGCAACTGTTGGCG – 3') that bind to drg internal sites and produce a 705 bp fragment in isolates containing the drg gene. PCR amplification was also performed using primer pairs Drg5F (5' – TGTCTAAAGAACTCAAAG – 3') / DrgB3 (5' – CGGTATCGAAAAATAAAT – 3') and Drg3F (5' – ATCCATCCAATTTCCCCA – 3') / DamB5 (5' – AAATGCCGTCTGAA – 3') based on dam and drg coding regions in order to confirm that dam inactivation was due to drg insertion. Amplicons corresponding to the drg gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously [46] and their nucleotide sequences determined on both strands by cycle sequencing. BigDye™ terminators (ABI, Foster city, California) and the same primers as used for amplification were employed in the extension reactions and the labelled reaction products separated on an ABI 3700 automated DNA sequencer. Each fragment was sequenced at least once on each strand and the sequences assembled with the STADEN suite of computer programs. Each unique drg sequence was assigned an arbitrary allele number in order of discovery. Data analysis Percentage G+C was calculated using the program START [47]. Split decomposition analysis of the drg gene fragments was performed using SPLITSTREE, version 3.1 [48]. The proportion of non-synonymous to synonymous substitutions was calculated by the method of Nei and Gojobori [49] using the program MEGA2 [50]. Authors' contributions KAJ carried out the nucleotide sequencing and analysis of the data. LS performed the PCR screening of the isolates. ERM and MCJM conceived of the study and participated in its design and coordination. Supplementary Material Additional File 1 Bacterial isolates used in the study Click here for file Acknowledegements MCJM is a Wellcome Trust Senior Research Fellow. This work was supported by an award from the Wellcome Trust to MCJM (grant 055104). Figures and Tables Figure 1 Variable sites in drg gene fragments Figure 2 Splits graph of drg gene fragments. The drg alleles found in the major drg+ hyper-invasive lineages are annotated. Table 1 Genetic variation in MLST and drg loci Locus No. of alleles (no./100 isolates) No. (%) of polymorphic sites dN/dS Average p-distance %G+C abcZ 15 (14) 75 (17.4) 0.05 0.052 51.1 adk 10 (9.4) 17 (3.7) 0.02 0.012 52.3 aroE 18 (16.8) 166 (34) 0.293 0.106 55.6 fumC 19 (17.8) 38 (8.2) 0.024 0.022 57.4 gdh 16 (15) 28 (5.6) 0.05 0.019 52.3 pdhC 24 (22.4) 80 (16.7) 0.07 0.054 55.9 pgm 21 (19.6) 77 (17) 0.121 0.050 54.2 drg 19 (31.1*) 48 (7.4) † 0.11 0.021 42.4 *only 61 isolates contained drg † excluding indels – length of fragment: 650 ==== Refs Broome CV The carrier state: Neisseria meningitidis. J Antimicrob Chemother 1986 18 25 34 3091563 Cartwright K Cartwright K Meningococcal carriage and disease Meningococcal disease 1995 Chichester: John Wiley & Sons Ltd 115 146 Bygraves JA Urwin R Fox AJ Gray SJ Russell JE Feavers IM Maiden MCJ Population genetic and evolutionary approaches to the analysis of Neisseria meningitidis isolates belonging to the ET-5 complex J Bacteriol 1999 181 5551 5556 10482493 Feavers IM Gray SJ Urwin R Russell JE Bygraves JA Kaczmarski EB Maiden MCJ Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak J Clin Microbiol 1999 37 3883 3887 10565901 Kriz P Giorgini D Musilek M Larribe M Taha MK Microevolution through DNA exchange among strains of Neisseria meningitidis isolated during an outbreak in the Czech Republic Res Microbiol 1999 150 273 80 10376489 10.1016/S0923-2508(99)80052-7 Feil EJ Enright MC Spratt BG Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae Res Microbiol 2000 151 465 9 10961460 10.1016/S0923-2508(00)00168-6 Feil EJ Maiden MCJ Achtman M Spratt BG The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis Mol Biol Evol 1999 16 1496 1502 10555280 Caugant DA Population genetics and molecular epidemiology of Neisseria meningitidis Apmis 1998 106 505 25 9674888 Caugant DA Mocca LF Frasch CE Frøholm LO Zollinger WD Selander RK Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern J Bacteriol 1987 169 2781 2792 3108242 Maiden MCJ Bygraves JA Feil E Morelli G Russell JE Urwin R Zhang Q Zhou J Zurth K Caugant DA Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms Proc Natl Acad Sci USA 1998 95 3140 3145 9501229 10.1073/pnas.95.6.3140 Modrich P Mechanisms and biological effects of mismatch repair Annu Rev Genet 1991 25 229 53 1812808 10.1146/annurev.ge.25.120191.001305 Moxon ER Rainey PB Nowak MA Lenski RE Adaptive evolution of highly mutable loci in pathogenic bacteria Curr Biol 1994 4 24 32 7922307 10.1016/S0960-9822(00)00005-1 Saunders NJ Jeffries AC Peden JF Hood DW Tettelin H Rappuoli R Moxon ER Repeat associated phase variable genes in the complete genome sequence of Neisseria meningitidis MC58 Mol Microbiol 2000 37 207 215 10931317 10.1046/j.1365-2958.2000.02000.x Hammerschmidt S Birkholz C Zahringer U Robertson BD van Putten J Ebeling O Frosch M Contribution of genes from the capsule gene complex cps to lipooligosaccharide biosynthesis and serum resistance in Neisseria meningitidis Mol Microbiol 1994 11 885 96 8022265 Vogel U Hammerschmidt S Frosch M Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitis serogroup B are prerequisites for virulence of meningococci in the infant rat Med Microbiol Immunol (Berl) 1996 185 81 7 8884739 10.1007/s004300050018 Stephens DS Spellman PA Swartley JS Effect of the (alpha 2–>8)-linked polysialic acid capsule on adherence of Neisseria meningitidis to human mucosal cells J Infect Dis 1993 167 475 479 8380612 Hammerschmidt S Muller A Sillmann H Muhlenhoff M Borrow R Fox A van Putten J Zollinger WD Gerardy-Schahn R Frosch M Capsule phase variation in Neisseria meningitidis serogroup B by slipped-strand mispairing in the polysialyltransferase gene siaD: correlation with bacterial invasion and the outbreak of meningococcal disease Mol Microbiol 1996 20 1211 1220 8809773 Virji M Makepeace K Ferguson DJP Achtman M Sarkari J Moxon ER Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis. Mol Microbiol 1992 6 2785 2795 1435257 Virji M Makepeace K Ferguson DJ Achtman M Moxon ER Meningococcal Opa and Opc proteins: their role in colonization and invasion of humanepithelial and endothelial cells Mol Microbiol 1993 10 499 510 7968528 Sarkari J Pandit N Moxon ER Achtman M Variable expression of the Opc outer membrane protein in Neisseria meningitidis is caused by size variation of a promotor containing poly-cytidine Mol Microbiol 1994 13 207 217 7984102 Virji M Alexandrescu C Ferguson DJ Saunders JR Moxon ER Variations in the expression of pili: the effect on adherence of Neisseria meningitidis tohuman epithelial and endothelial cells Mol Microbiol 1992 6 1271 1279 1353602 van der Ende A Hopman CTP Dankert J Multiple mechanisms of phase variation of PorA in Neisseria meningitidis Infect Immun 2000 68 6685 6690 11083782 10.1128/IAI.68.12.6685-6690.2000 Dehio C Gray-Owen SD Meyer TF The role of neisserial Opa proteins in interactions with host cells Trends Microbiol 1998 6 489 494 10036728 10.1016/S0966-842X(98)01365-1 Garcia-Del Portillo F Pucciarelli MG Casadesus J DNA adenine methylase mutants of Salmonella typhimurium show defects in protein secretion, cell invasion, and M cell cytotoxicity Proc Natl Acad Sci USA 1999 96 11578 83 10500219 10.1073/pnas.96.20.11578 Watson ME JrJarisch J Smith AL Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence Mol Microbiol 2004 53 651 64 15228541 10.1111/j.1365-2958.2004.04140.x Low DA Weyand NJ Mahan MJ Roles of DNA adenine methylation in regulating bacterial gene expression and virulence Infect Immun 2001 69 7197 204 11705888 10.1128/IAI.69.12.7197-7204.2001 Bucci C Lavitola A Salvatore P Del Giudice L Massardo DR Bruni CB Alifano P Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype Mol Cell 1999 3 435 45 10230396 10.1016/S1097-2765(00)80471-2 Richardson AR Stojiljkovic I Mismatch repair and the regulation of phase variation in Neisseria meningitidis Mol Microbiol 2001 40 645 55 11359570 10.1046/j.1365-2958.2001.02408.x Martin P Sun L Hood DW Moxon ER Involvement of genes of genome maintenance in the regulation of phase variation frequencies in Neisseria meningitidis Microbiology 2004 Cantalupo G Bucci C Salvatore P Pagliarulo C Roberti V Lavitola A Bruni CB Alifano P Evolution and function of the neisserial dam-replacing gene FEBS Lett 2001 495 178 83 11334887 10.1016/S0014-5793(01)02388-2 Lacks S Greenberg B A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA J Biol Chem 1975 250 4060 66 236309 Levin BR The evolution and maintenance of virulence in microparasites Emerg Infect Dis 1996 2 93 102 8903208 Maiden MCJ High-throughput sequencing in the population analysis of bacterial pathogens Int J Med Microbiol 2000 290 183 190 11045923 Tinsley CR Nassif X Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related bacteria expressing two different pathogenicities Proc Natl Acad Sci U S A 1996 93 11109 14 8855317 10.1073/pnas.93.20.11109 Peltola H Meningococcal disease: still with us Rev Infect Dis 1983 5 71 91 6338571 Waldron DE Owen P Dorman CJ Competitive interaction of the OxyR DNA-binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli Mol Microbiol 2002 44 509 20 11972787 10.1046/j.1365-2958.2002.02905.x Caugant DA Frøholm LO Bovre K Holten E Frasch CE Mocca LF Zollinger WD Selander RK Intercontinental spread of Neisseria meningitidis clones of the ET-5 complex Antonie Leeuwenhoek J Microbiol 1987 53 389 394 Wang J-F Caugant DA Li X Hu X Poolman JT Crowe BA Achtman M Clonal and antigenic analysis of serogroup A Neisseria meningitidis with particular reference to epidemiological features of epidemic meningitis in China Infect Immun 1992 60 5267 5282 1452360 Wang J-F Caugant DA Morelli G Koumaré B Achtman M Antigenic and epidemiological properties of the ET-37 complex of Neisseria meningitidis J Infect Dis 1993 167 1320 1329 8501321 Jelfs J Munro R Ashto FE Caugant DA Genetic characterization of a new variant within the ET-37 complex of Neisseria meningitidis associated with outbreaks in various parts of the world Epidemiol Infect 2000 125 285 98 11117951 10.1017/S0950268899004471 Holmes EC Urwin R Maiden MCJ The influence of recombination on the population structure and evolution of the human pathogen Neisseria meningitidis Mol Biol Evol 1999 16 741 749 10368953 Ritchot N Roy PH DNA methylation in Neisseria gonorrhoeae and other Neisseriae Gene 1990 86 103 6 2155857 10.1016/0378-1119(90)90120-G Claus H Friedrich A Frosch M Vogel U Differential distribution of novel restriction-modification systems in clonal lineages of Neisseria meningitidis J Bacteriol 2000 182 1296 303 10671450 10.1128/JB.182.5.1296-1303.2000 Bart A Dankert J van der Ende A Representational difference analysis of Neisseria meningitidis identifies sequences that are specific for the hyper-virulent lineage III clone FEMS Microbiol Lett 2000 188 111 4 10913691 10.1016/S0378-1097(00)00221-4 Claus H Stoevesandt J Frosch M Vogel U Genetic isolation of meningococci of the electrophoretic type 37 complex J Bacteriol 2001 183 2570 2575 11274117 10.1128/JB.183.8.2570-2575.2001 Embley TM The linear PCR reaction: a simple and robust method for sequencing amplified rRNA genes Lett Appl Microbiol 1991 13 171 174 1370053 Jolley KA Feil EJ Chan MS Maiden MC Sequence type analysis and recombinational tests (START) Bioinformatics 2001 17 1230 1 11751234 10.1093/bioinformatics/17.12.1230 Huson DH SplitsTree: analyzing and visualizing evolutionary data Bioinformatics 1998 14 68 73 9520503 10.1093/bioinformatics/14.1.68 Nei M Gojobori T Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions Mol Biol Evol 1986 3 418 26 3444411 Kumar S Tamura K Jakobsen IB Nei M MEGA2: molecular evolutionary genetics analysis software Bioinformatics 2001 17 1244 5 11751241 10.1093/bioinformatics/17.12.1244

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==== Front BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-141533314010.1186/1471-2199-5-14Research ArticleSequence periodicity of Escherichia coli is concentrated in intergenic regions Hosid Sergey [email protected] Edward N [email protected] Alexander [email protected] Genome Diversity Center, Institute of Evolution, University of Haifa, Mt. Carmel 31905 ISRAEL2004 26 8 2004 5 14 14 31 12 2003 26 8 2004 Copyright © 2004 Hosid et al; licensee BioMed Central Ltd.2004Hosid et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Sequence periodicity with a period close to the DNA helical repeat is a very basic genomic property. This genomic feature was demonstrated for many prokaryotic genomes. The Escherichia coli sequences display the period close to 11 base pairs. Results Here we demonstrate that practically only ApA/TpT dinucleotides contribute to overall dinucleotide periodicity in Escherichia coli. The noncoding sequences reveal this periodicity much more prominently compared to protein-coding sequences. The sequence periodicity of ApC/GpT, ApT and GpC dinucleotides along the Escherichia coli K-12 is found to be located as well mainly within the intergenic regions. Conclusions The observed concentration of the dinucleotide sequence periodicity in the intergenic regions of E. coli suggests that the periodicity is a typical property of prokaryotic intergenic regions. We suppose that this preferential distribution of dinucleotide periodicity serves many biological functions; first of all, the regulation of transcription. ==== Body Background DNA sequence periodicity with the period about 10–11 base pairs (bp) has been long known in eukaryotic DNA sequences. It was discovered recently in prokaryotic sequences as well [1-6]. The periodicity in Eubacteria sequences usually shows the period close to 11 bp [1]. This period is clearly different from the structural helical period of 10.5–10.6 bp/turn [7,8]. The difference was interpreted [1,2] as a possible reflection of the sequence dependent writhe of prokaryotic DNA. In the work [9] it was demonstrated that the periodicity in the bacterial genomes, in E. coli as well, is distributed in a non-uniform way, in scattered segments of the size 100–150 bases. It was also known for a long time that quite a few DNA promoter regions of E. coli possess the sequence periodicity of AA and TT dinucleotides [10]. The sequence periodicity of AA/TT dinucleotides is frequently associated with sequence-dependent DNA curvature, which is known to play an important role in the initiation of transcription of many genes (for reviews, see [11-15]). Using different models and approaches for prediction of intrinsic DNA curvature it was shown that many E. coli promoters have upstream curved sequences [16,17]. Pedersen et al. [18] showed that promoter area frequently has an unusual sequence structure. This region possesses higher DNA curvature, more rigid and less stable. Moreover, in our study of prokaryotic terminators of transcription (Hosid and Bolshoy, submitted) we have found that in E. coli DNA curvature peaks are frequently located downstream of the CDS. Since the dinucleotide periodicity with the period close to the helical repeat is associated with DNA intrinsic curvature [19-23], the curvature distribution along DNA would suggest similar distribution of DNA sequence periodicity. In this work, the sequence dinucleotide periodicity in E. coli and its distribution along the genome are systematically analyzed. A strong preference of intergenic regions to express the sequence periodicity of AA, AC, GC, and TT dinucleotides is discovered. Results and Discussion Positional autocorrelation analysis of the nucleotide sequences is an appropriate tool to detect all major characteristic distances in the sequences, the periodicities in particular. The complete genome of E. coli, as well as its coding and noncoding regions, was subjected to this procedure. Resulting autocorrelation profiles for all 16 dinucleotides (data not shown) were further analyzed by Fourier transform. In Fig. 1 the corresponding spectra are shown. The analysis demonstrates presence of the sequence periodicity of AA and TT dinucleotides with a period close to 11 bp mostly in intergenic regions, and weaker periodicity of AC and GC notably exclusively in intergenic regions. All 16 dinucleotides show periodicity of 3 bp, a well-known characteristics of the coding sequences, e.g. [24,25]. Weak 2 bp periodicity of AT and TA is also observed in intergenic regions. It indicates, perhaps, presence of tandem ApT repeats. A weak 10 bp periodicity of GC in intergenic regions, probably, corresponds to terminator regions (work in progress). The amplitudes of the 11 bp periodicity of AA and TT are the highest, even comparable with 3 bp coding periodicity. We, thus, focused on AA and TT distributions. Figure 1 Periodograms of the distance distributions of 16 dinucleotides in E. coli genome. The complete nucleotide sequence of E. coli, as well as subsets of its coding and noncoding regions, was subjected to the positional autocorrelation analysis for all 16 dinucleotides separately. Resulting autocorrelation profiles were after that analyzed by Fourier transform. The black lines correspond to the whole genome, the blue curves – to the coding sequences, and the red curves – to the noncoding sequences. To screen the genome of E. coli and find out where the periodical regions are located, we chose the period 11.2 bp [1,2,5] and this study (Fig. 1); and the window of 150 bp [9,26]. We used periodical AA and TT probes with the above periodicity to correlate with the E. coli genome sequence and to detect the periodical sites. This calculation shows that the periodicity is not evenly distributed along the E. coli genome. In Fig. 2, the typical maps for several large segments of the E. coli genome are shown. The periodicity is distinctly located in certain regions. Many of the peaks observed are found to correspond to the intergenic regions (indicated by the black bars at the top). For example, two such peaks of periodicity in Fig. 2a correspond to the intergenic regions. Three such maxima are observed in Fig. 2b, three in Fig. 2c, and two in Fig. 2d. For the genome sections in Fig. 2 about 2/3 of the intergenic regions are associated with the local periodicity. Figure 2 Four examples of periodicity maps for fragments of E. coli genome. The maps were smoothed by running average with window 51 bp. The black bars on the top of the plot correspond to positions of intergenic regions. To verify the apparent strong correlation between the intergenic regions and AA/TT periodicity, we split intergenic regions in several families by size and analyzed the subsets separately by aligning (centering) the regions and summing up the respective local periodicity distributions. The combined maps for intergenic regions with a size from 50 to 150 bp, from 150 to 250 bp, from 250 to 350 bp, from 350 to 450 bp, and from 450 to 550 bp are shown in Fig. 3. This figure demonstrates, indeed, that intergenic regions are typically periodic, irrespective of the size. The average amplitudes of the observed periodicities – 0.1–0.25 units – are comparable with the amplitudes in Fig. 2, which indicates that, indeed a large proportion of the intergenic regions are periodical. Figure 3 The averaged maps of periodicity are synchronized at the centers of intergenic regions and smoothed by a running average of 51 bp. Five families of the intergenic regions with different lengths are presented: a) 100 ± 50, bp 1073 sequences, b) 200 ± 50 bp, 602 sequences, c) 300 ± 50 bp, 319 sequences, d) 400 ± 50 bp, 160 sequences, and e) 500 ± 50 bp, 78 sequences. The black bars at the bottom of the each figure correspond to the average intergenic region. The gray bands around black dashed lines correspond to standard deviations around randomized background. To verify the choice of the period 11.2 bases, we calculated the periodicity maps for highly populated group of the regions of the size 200 ± 50 bp, by assuming different periods in the range 10.5–12.5 bases. The resonance 3D plot in Fig. 4 indicates that the best-fit period is 11.3 ± 0.4 bp, which confirms earlier estimates of the E. coli DNA sequence periodicity. Figure 4 The 3D resonance plot for the intergenic regions of length 200 ± 50 bp. The maximum of resonance the plot corresponds to period 11.3 ± 0.4 bp. The contour around the maximum is also shown as a projection at the base line level. The spectral analysis (Fig. 1) and examples of the periodicity distribution maps (Fig. 2) show that apart from described correlation among the intergenic regions and AA/TT periodicity, there are numerous sites of periodicity located within coding sequences. Work is in progress to find out the functional relevance, if any, of these sites. Conclusions The observed concentration of the sequence periodicity in the intergenic regions corroborates earlier results and suggests that the periodicity is a typical property of the intergenic regions. Methods Genome data The sequence of the whole genome of Escherichia coli K-12 MG1655, locus U00096, 4639221 base pairs, was taken from the National Center of Biotechnology Information . Intergenic regions were identified in accordance with the annotation to this genome of E. coli and gathered in a separate dataset. Fourier transform of positional autocorrelation function Autocorrelation profile X was calculated for each dinucleotide separately. For the calculation of ApA autocorrelation, for example, we calculated the number of occurrences of pairs ApA – ApA in a distance k, and designated it by Xk. Spectral analysis of autocorrelation profile X was obtained using the following formulae: where fp is normalized wave-function amplitude of period p, X is an autocorrelation profile for one chosen dinucleotide, Xi is its value in position i, is its average value, and W is a maximal considered autocorrelation distance (in our case 100 bp). Sequence periodicity As a probe of periodicity the sine waves with period T were taken to describe idealized periodical distribution of AA and TT dinucleotides within window W. The probes were correlated with E. coli sequences by moving the probes along the sequences and calculating the value C for every position. where i is an index of a dinucleotide position in the window W and The value Cmax is introduced for the normalization purposes. It is calculated as follows: where i is a position in the window W and Ideally periodical sequence segments would be, therefore, described by C = 1, while segments with no periodicity would correspond to C = 0. The results of these calculations are presented as maps of the sequence periodicity. The four sample maps are shown in Fig. 2a,2b,2c,2d. Synchronization of the maps The maps around intergenic regions were combined (summed) separately for the groups of similar sizes of the intergenic regions. Five such groups were analyzed: 100 ± 50 bp, 200 ± 50 bp, 300 ± 50 bp, 400 ± 50 bp, and 500 ± 50 bp. For each group the maps were synchronized at the respective intergenic centers and the sums of the maps were calculated and smoothed by a running average within 51 bp. The standard deviations for the combined plots were estimated by generating random sequences of the same size and dinucleotides composition for each group separately and averaging the respective periodicity maps. The resonance plot The resonance 3D plot for the intergenic regions of length 200 ± 50 bp was built from calculations with different periods T in the interval 10–12.5 bp. One-third (202) of the most periodic maps of this group was taken for the calculation. The maps for different periods T were smoothed five times by a running average over 51 bp. The baselines were set to 0. The surface of 3D plot was smoothed 3 times by a running average over 9 point square elements, on the grid with separations 0.1 bp for T, and 20 bp for sequence position. Competing interests None declared. Authors' contributions SH carried out all graphics. ENT and AB participated in the design of the study and analysis of results. All authors drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments We thank V. Kirzhner and all members of the Genome Diversity Center for fruitful discussions and critical comments on the paper. A.B. is grateful to Professors T. Ratiu and J.H. Maddocks from the Bernoulli Institute at the Swiss Federal Institute of Technology for the kind invitation to visit "Centre Bernoulli" for two months. S.H. and A.B. are partially supported by the FIRST Foundation of the Israel Academy of Science and Humanities. ==== Refs Herzel H Weiss O Trifonov EN Sequence periodicity in complete genomes of Archaea suggests positive supercoiling J Biomol Struct Dyn 1998 16 341 345 9833672 Herzel H Weiss O Trifonov EN 10–11 bp periodicities in complete genomes reflect protein structure and DNA folding Bioinformatics 1999 15 187 193 10222405 10.1093/bioinformatics/15.3.187 Ozoline ON Deev AA Trifonov EN DNA bendability – a novel feature in E-coli promoter recognition J Biomol Struct Dyn 1999 16 825 831 10217452 Tomita M Wada M Kawashima Y ApA dinucleotide periodicity in prokaryote, eukaryote, and organelle genomes J Mol Evol 1999 49 182 192 10441670 Worning P Jensen LJ Nelson KE Brunak S Ussery DW Structural analysis of DNA sequence: evidence for lateral gene transfer in Thermotoga maritima Nucleic Acids Res 2000 28 706 709 10637321 10.1093/nar/28.3.706 Petersen L Larsen TS Ussery DW On SLW Krogh A RpoD promoters in Campylobacter jejuni exhibit a strong periodic signal instead of a-35 box J Mol Biol 2003 326 1361 1372 12595250 10.1016/S0022-2836(03)00034-2 Strauss F Gaillard C Prunell A Helical Periodicity of DNA, Poly(Da) . Poly(Dt) and Poly(Da-Dt) . Poly(Da-Dt) in Solution Eur J Biochem 1981 118 215 222 7285918 Peck LJ Wang JC Sequence Dependence of the Helical Repeat of DNA in Solution Nature 1981 292 375 378 6265793 Tolstorukov MY Virnik K Adhya S Zhurkin VB Genome-wide A-tract distribution and DNA packaging in pro-and eukaryotes [abstract] J Biomol Struct Dyn 2003 20 869 870 Plaskon RR Wartell RM Sequence Distributions Associated with DNA Curvature Are Found Upstream of Strong Escherichia-Coli Promoters Nucleic Acids Res 1987 15 785 796 3547329 Trifonov EN Curved DNA Crc Critical Reviews in Biochemistry 1985 19 89 106 3905255 Hagerman PJ Sequence-directed curvature of DNA Annu Rev Biochem 1990 59 755 781 2197990 10.1146/annurev.bi.59.070190.003543 Harrington RE DNA Curving and Bending in Protein DNA Recognition Mol Microbiol 1992 6 2549 2555 1333034 Perez-Martin J Rojo F de Lorenzo V Promoters responsive to DNA bending: a common theme in prokaryotic gene expression Microbiol Rev 1994 58 268 290 8078436 Gabrielian A Pongor S Correlation of intrinsic DNA curvature with DNA property periodicity FEBS Lett 1996 393 65 68 8804425 10.1016/0014-5793(96)00855-1 Lisser S Margalit H Determination of Common Structural Features in Escherichia-Coli Promoters by Computer-Analysis Eur J Biochem 1994 223 823 830 8055959 Gabrielian AE Landsman D Bolshoy A Curved DNA in promoter sequences In Silico Biol 2000 1 183 196 Pedersen AG Jensen LJ Brunak S Staerfeldt HH Ussery DW A DNA structural atlas for Escherichia coli J Mol Biol 2000 299 907 930 10843847 10.1006/jmbi.2000.3787 Marini JC Levene SD Crothers DM Englund PT Bent Helical Structure in Kinetoplast DNA Proc Natl Acad Sci U S A 1982 79 7664 7668 16593261 Hagerman PJ Evidence for the existence of stable curvature of DNA in solution Proc Natl Acad Sci U S A 1984 81 4632 4636 6087336 Hagerman PJ Sequence dependence of the curvature of DNA – a test of the phasing hypothesis Biochemistry 1985 24 7033 7037 4084556 Koo HS Wu HM Crothers DM DNA bending at adenine . thymine tracts Nature 1986 320 501 506 3960133 Ulanovsky L Bodner M Trifonov EN Choder M Curved DNA – Design, Synthesis, and Circularization Proc Natl Acad Sci U S A 1986 83 862 866 3456570 Trifonov EN Translation Framing Code and Frame-Monitoring Mechanism as Suggested by the Analysis of Messenger-RNA and 16 S Ribosomal-RNA Nucleotide-Sequences J Mol Biol 1987 194 643 652 2443708 Trifonov EN 3-, 10.5-, 200-and 400-base periodicities in genome sequences Physica A 1998 249 511 516 10.1016/S0378-4371(97)00510-4 Bolshoy A Nevo E Ecologic genomics of DNA: upstream bending in prokaryotic promoters Genome Res 2000 10 1185 1193 10958636 10.1101/gr.10.8.1185

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-291533313910.1186/1471-2202-5-29Research ArticleSpontaneous activity of rat pretectal nuclear complex neurons in vitro Prochnow Nora [email protected] Matthias [email protected] Allgemeine Zoologie & Neurobiologie, Ruhr-Universität Bochum, 44780 Bochum, Germany2004 27 8 2004 5 29 29 24 6 2004 27 8 2004 Copyright © 2004 Prochnow and Schmidt; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Neurons in the mammalian pretectum are involved in the control of various visual and oculomotor tasks. Because functionally independent pretectal cell populations show a wide variation of response types to visual stimulation in vivo, they may also differ in their intrinsic properties when recorded in vitro. We therefore performed whole-cell patch clamp recordings from neurons in the caudal third of the pretectal nuclear complex in frontal brain slices obtained from 3 to 6 week old hooded rats and tried to classify pretectal neurons electrophysiologically. Results Pretectal neurons showed various response types to intracellular depolarizations, including bursting and regular firing behavior. One population of pretectal nuclear complex neurons could be particularly distinguished from others because they displayed spontaneous activity in vitro. These cells had more positive resting potentials and higher input resistances than cells that were not spontaneously active. The maintained firing of spontaneously active pretectal cells was characterized by only small variances in interspike intervals and thus showed a regular temporal patterning. The firing rate was directly correlated to the membrane potential. Removing excitatory inputs by blockade of AMPA and/or NMDA receptors did not change the spontaneous activity. Simultaneous blockade of excitatory and inhibitory synaptic input by a substitution of extracellular calcium with cobalt neither changed the firing rate nor its temporal patterning. Each action potential was preceeded by a depolarizing inward current which was insensitive to calcium removal but which disappeared in the presence of tetrodotoxin. Conclusions Our results indicate that a specific subpopulation of pretectal neurons is capable of generating maintained activity in the absence of any external synaptic input. This maintained activity depends on a sodium conductance and is independent from calcium currents. ==== Body Background Neurons in the mammalian pretectal nuclear complex (PNC) are involved in the control of various oculomotor reflexes, like the pupillary light reflex and the optokinetic reflex (OKR). Pupil constriction is controlled by neurons in the olivary pretectal nucleus that project bilaterally to the Edinger-Westphal nucleus [1-7]. Slow eye movements during OKR are generated by neurons in the nucleus of the optic tract (NOT) and in the adjacent dorsal terminal nucleus (DTN) of the accessory optic system (AOS) which project to the inferior olive (IO) and the nucleus prepositus hypoglossi [8-13]. In addition, PNC neurons carry signals related to saccadic eye movements to the dorsal lateral geniculate nucleus (LGNd) [14-16] and to the extrageniculate thalamus [17]. Other, reciprocal, projections connect the PNC to its contralateral counterpart, to the ipsilateral superior colliculus, and to the other AOS nuclei. The functions of those projections, however, are still under debate [18-23]. Each projection target receives input from an independent PNC neuronal population. Therefore, multiple retrograde tracing, e.g. from contralateral PNC and IO [24], or LGNd and Pulvinar [25], does not double label PNC cells. Furthermore, neurons with different projection targets show different response properties in vivo. Thus, neurons involved in the pupillary light reflex respond tonically to the overall retinal luminance [1,3,6,26]. OKR-related neurons are directionally selective in response to slow movements of large visual stimuli [8,27-33]. Finally, PNC neurons that project to thalamic visual centers only respond to fast moving visual stimuli without directional selectivity [15,17,25,34-36]. Furthermore, activity patterns of visual responses also differ significantly between PNC cell populations. Thus, saccade-related PNC neurons show short high frequency activity bursts, while luminance neurons or OKR-related neurons exhibit tonic activity at moderate firing levels. Although such differences to some extent directly reflect the response properties of specific input systems, different intracellular properties might enforce activity patterns provided by different input systems. We therefore studied intrinsic properties of rat PNC cells in vitro. In particular, we describe a population of cells in the caudo-lateral PNC that is characterized by intrinsically generated spontaneous activity in vitro, which is an unusual property for neurons in a sensory relay structure. Results In total, we obtained whole-cell recordings from 114 pretectal nuclear complex (PNC) neurons. Slices included the caudal part of the pretectum (Fig. 1), cells were recorded from the NOT, the posterior pretectal nucleus (PPN), and the olivary pretectal nucleus (OPN). Depolarizing current injections induced various spike patterns, like bursting (Fig. 2A,2B,2C), non-adapting regular spiking (Fig. 2D), or irregular spiking (Fig. 2E). Usually, increasing the current amplitude also increased the firing rate, however, in about 31% (n = 35) of the cells, the firing rate showed a clear maximum in response to intermediate depolarizing current injections and decreased upon further depolarization (Fig. 2C). Input resistances ranged from 201.0 to 776.3 MΩ (mean 410 ± 166.5 MΩ), resting potentials varied between -41.0 and -74.3 mV (mean -54.6 ± 8.5 mV). All cells tested, irrespective of the response type, responded to OT stimulation. Characteristics of spontaneous activity Of the cells recorded, 73 PNC neurons showed spontaneous firing at resting potential. Camera lucida reconstructions revealed that these cells were characterized by large fusiform cell bodies (diam. 15 μm and above) and multipolar dendritic trees that did not show any preference in their orientation. Whenever axons were also visible, they could be followed to leave the pretectal area in a ventro-lateral direction which indicates that these cells project to extrapretectal targets (arrowheads in Fig. 3A). Their dendritic morphologies, however, did not allow to distinguish spontaneously active cells from PNC neurons that were not spontaneously active. Physiologically, all spontaneously active PNC neurons were characterized by a regular firing pattern when recorded at resting potential without any injected current (Fig. 3C). The firing rate at resting potential of spontaneously active PNC cells varied between 0.9 and 9.4 imp/s (mean 3.0 ± 2.1 imp/s). Depolarizing current injections induced tonic firing patterns with only marginal adaptation (Fig. 3D). Responses to hyperpolarizing current injections showed no sign of inward rectification. Furthermore, following cessation of hyperpolarizing current steps we never observed rebound spikes. Spontaneously active PNC cells on average had significantly higher input resistances (mean 454.1 ± 164.7 MΩ, p < 0.001), more positive resting potentials (mean -50.4 ± 7.0 mV, p < 0.001) and lower spike thresholds (mean -55.0 ± 3.96 mV, p < 0.001) than cells that did not show spontaneous activity (331.44 ± 137.1 MΩ, -58.4 ± 8.0 mV, and -40.66 ± 6.44 mV, respectively). In order to characterize the spike adaptation behavior of spontaneously active PNC cells, the holding potentials were increased in 5 mV steps by appropriate current injections in all recorded cells. In response to these depolarization steps, cells showed tonic increases of their firing rate without any sign of firing rate adaptation (Fig. 4). Also, no phasic firing rate increases were observed following the depolarizations. As could be already derived from current injections, the firing rate was directly correlated with the membrane potential. Increasing the membrane potential by positive current injections increased the firing rate until a maximum level was reached that could not be exceeded by further depolarization (Fig. 5A). Consequently, when the firing rate is plotted against the membrane potential, the course of the resulting function is sigmoidal (Fig. 5B). In order to get an impression about the regularity of the firing of spontaneously active PNC cells, interspike intervals (ISI) during maintained firing were analyzed in more detail (Fig. 6). Thus, maintained firing was recorded over a 10 s period at different membrane potentials and ISI histograms were generated from the recorded activity. ISIs obtained from these recordings followed a narrow unimodal Gaussian distribution with only little variation (Fig. 6B). According to the correlation between the firing rate and the membrane potential, depolarization of the cells resulted in shifts of the maximum of the Gaussian distribution towards lower ISI values. Depolarization, however, did not change the shape of the distribution. The regularity of the maintained firing of spontaneously active PNC cells is also supported by autocorrelograms of the recorded spike trains (Fig. 6C). The appearance of multiple equally spaced peaks in the autocorrelogram results from the regular timing of single spikes. Generation of spontaneous activity in vitro In order to test whether the spontaneous activity of PNC neurons in vitro depends on excitatory input, we first suppressed glutamatergic synaptic transmission and pharmacologically blocked AMPA receptors in 13 spontaneously active PNC cells (Fig. 7). As a control for the effectiveness of AMPA receptor blockade, the influence of the AMPA receptor antagonist CNQX on postsynaptic responses was monitored. In all cells tested, bath application of 20 μM CNQX resulted in a complete loss of EPSCs after electrical stimulation of optic tract afferent fibers (Fig. 7A,7E). Although excitatory input was obviously blocked by CNQX application, the maintained firing remained unchanged (Fig. 7B,7F). In particular, no drop in the firing rate was observed that could have been induced by a possible loss of excitatory input. Furthermore, the comparison of both the ISI distribution (Fig. 7C,7G) and the autocorrelograms (Fig. 7D,7H) obtained from spike trains before and during CNQX application did not show any significant difference. Hence, both the generation of spontaneous activity and its patterning seem to be independent from excitatory input via AMPA receptors. Similar results were achieved when NMDA receptors were blocked by bath application of 50 μM APV or when 2 mM kynurenic acid was applied to simultaneously block AMPA and NMDA receptors (N = 19). After having excluded glutamatergic synaptic inputs as a trigger for maintained firing, we tried to remove all synaptic input by adding cobalt to the extracellular medium in 12 spontaneously active PNC cells. This blocks the influx of calcium into the presynaptic terminal and thus prevents vesicular neurotransmitter release. Adding 1.5 mM CoCl2 to the bath completely suppressed all electrically evoked postsynaptic currents (Fig. 8A,8D) in all cells tested. In contrast to the complete loss of postsynaptic currents, however, the maintained firing always remained unchanged (Fig. 8B,8E). As during glutamate receptor blockade, no reduction of the firing rate was observed that could have indicated the removal of an excitatory input. In addition, no increase of the firing rate appeared that could have indicated a loss in tonic inhibitory input regulating maintained activity. Finally, examination of the ISI distribution in the spike trains demonstrated that the patterning of the maintained activity also did not show any significant difference in the presence of Cobalt (Fig. 8C,8F). This indicates that spontaneously active PNC cells generate their firing intrinsically without any external synaptic input. In current-clamp mode, each action potential was preceded by a depolarizing ramp (see, for example, Figs. 3C and 4). When cells were hyperpolarized to membrane potentials just below their resting potential single depolarizing ramps appeared that were not followed by an action potential. Concomitantly, in voltage-clamp mode, each unclamped action potential was preceded by an depolarizing inward current (Fig. 9A). Because they did not disappear after substitution of calcium by cobalt in the external solution these current ramps were calcium independent. However, when 1 μM tetrodotoxin (TTX), a selective blocker of sodium channels, was added to the bath solution current ramps were eliminated together with the action potentials (Fig. 9B,9C) in all seven cells tested. Discussion We have examined neurons in the rat PNC that are characterized by maintained activity in vitro. These spontaneously active PNC cells do not differ in their dendritic morphology from PNC cells that are not spontaneously active, but they show higher input resistances, more positive resting potentials, and lower spike thresholds. Furthermore, our results indicate that, firstly, all PNC cells that display spontaneous activity share firing characteristics, such as very regularly patterned spike trains and pure tonic firing rate increases in response to intracellular depolarizations. Secondly, the generation of the maintained firing of these cells is independent from excitatory synaptic input which suggests that these cells exhibit specific intrinsic properties that underly the generation of spontaneous activity. Finally, the patterning of the maintained firing is also independent from synaptic input, both excitatory and inhibitory, which indicates that their intrinsic membrane properties determine the firing pattern. To our knowledge, this is the first demonstration of spontaneous activity generated in vitro by cells in a subcortical visual relay structure. Generation of spontaneous activity in vitro Neurons that show spontaneous activity in vitro have been reported to exist in various mammalian CNS structures. Most extensively studied, spontaneously active neurons exist in the suprachiasmatic nucleus (SCN) which contains the biological clock that generates circadian rhythmicity. Thus, SCN cells not only generate spontaneous activity in vitro but they also maintain their circadian firing pattern [37-39]. Other populations of cells spontaneously active in vitro are the cholinergic interneurons in the neostriatum [40], dopaminergic cells in the substantia nigra [41,42], neurons in the subthalamic nucleus [43,44], neurons in the medial vestibular nucleus [45-47], cells from deep cerebellar nuclei [48,49], and cerebellar Purkinje cells [48,50,51]. Within the visual system, particularly the subcortical portion, spontaneous activity in vitro has been described to occur in thalamocortical neurons [52] and in isolated dopanimergic cells from the retina [53]. However, in the mammalian PNC cells that show maintained activity in vitro have not yet been reported. These cells are characterized by very regular firing pattern and monotonically increasing firing rates in response to intracellular depolarization. They differed from PNC neurons not spontaneously active by higher input resistance and more positive resting membrane potentials. Because glutamate receptor blockade did not change the firing characteristics, neither the firing rate nor the patterning of the firing, excitatory synaptic input through glutamate receptors seems unnecessary for the generation of the spontaneous activity. Furthermore, maintaining spontaneous firing in these PNC neurons also seems to be independent from synaptic input through other neurotransmitter systems because blockade of synaptic transmission by bath application of Cobalt did not change the firing. Thus, we conclude that the firing pattern is neither shaped by tonic excitation nor by tonic inhibition. Similar to spontaneously active cells in the structures noted above, our PNC neurons must possess intrinsic membrane properties that allow the generation of maintained activity. As far as the ionic mechanisms underlying the generation of spontaneous activity are concerned our results suggest that it critically depends on a TTX-sensitive sodium conductance. This sodium conductance leads to a steady inward current following spike afterhyperpolarization which induces membrane depolarization to spike threshold. This is similar to the ionic mechanism that is responsible for spike generation in spontaneously active neurons in the suprachiasmatic nucleus [54]. Because spontaneous firing in PNC neurons was unchanged by calcium substitution with cobalt the spontaneous activity generation seems independent from calcium conductances. Possible functional implications A characteristic response property of all spontaneously active PNC cells was that the firing rate increases to depolarizing voltage steps did not show any phasic components. This makes these cells perfectly suited to code maintained or tonic neuronal information. Of course, one has to keep in mind that neuronal response properties in vivo are shaped by numeous afferent input systems most of which are absent in the slice preparation. Thus, tonic inhibitory input could mask the maintained firing of spontaneously active PNC cells leading to a very different response pattern in vivo. Consequently, the maintained firing might become apparent only under very specific stimulus conditions upon withdrawal of the inhibitory input. However, from a functionl point of view, we regard it more reasonable to assume that PNC cells which are spontaneously active in vitro also exhibit tonic firing in vivo. Reviewing the known functions served by PNC neurons in vivo reveals only few reasonable suggestions for the possible functions which spontaneously active cells might accomplish. Thus, cells that typically show sustained activity in vivo are involved in the pupillary light reflex [2,3,6,26,55]. These cells are characterized by tonic increases of their firing rate to increases in the background luminance. However, these cells are predominantly found in the olivary pretectal nucleus (OPN) which is located in the rostro-medial PNC [reviewed in [56]]. Because our recordings were topographically restricted to the caudo-lateral PNC, particularly to the nucleus of the optic tract (NOT) and the posterior pretectal nucleus (PPN), it seems unlikely that we recorded from luminance neurons in the OPN. Neurons found in NOT and PPN include various functional cell populations. One of them has been associated with the generation of slow phase eye movements during OKR while others seem to transfer visual information linked to the execution of saccadic eye movements. Cells from these latter populations are all characterized by short duration, high frequency burst responses to fast image motions or rapid eye movements [15,17,25,34-36,57] and thus seem unlikely to correlate with cells that show maintained activity in vitro. Furthermore, because the timing of postsynaptic spikes with respect to their presynaptic input might be of considerable functional importance for saccade-related neurons such cells should exhibit lower input resistance than neurons for which spike time precision is less important. Low input resistances allow faster depolarization of the postsynaptic membrane and, hence, less temporal variance or "jitter" between presynaptic and postsynaptic spikes will occur. However, spontaneously active PNC cells on average showed higher input resistances in our sample and we therefore do not think that they represent saccade-related PNC neurons. On the other hand, cells that control compensatory eye movements during OKN are characterized by tonic firing in vivo when appropriately stimulated by low speed horizontal movements of whole field visual stimuli. In all mammals studied, neurons in the right PNC are excited by rightward stimulus motion and control eye movements to the right, while neurons in the left PNC are activated by leftward stimulus motion and control eye movements to the left [8,27-33]. The response properties of OKN-related PNC neurons to a large extent reflect response characteristics of their retinal afferents which are also activated by slow stimulus movements and show strong directional selectivity [58]. However, it may be functionally important to assure a constant level of maintained activity in OKN-related PNC neurons by additional intrinsic mechanisms in the absence of appropriate visual stimuli. Unilateral inactivation of PNC neuronal activity by focal injections of muscimol or lidocaine leads to spontaneous eye movements in darkness [11,59]. Because of the directional specificity in the PNC, inactivation of the right PNC elicits eye movements to the left, while inactivation of the left PNC elicits eye movements to the right. Thus, eye movements that appear after PNC inactivation seem to result from a distortion of a balanced activity between the two PNCs. Whenever the balance is distorted, premotor target structures postsynaptic to OKR-related PNC neurons receive stronger input from the PNC of one side and eye movements are elicited accordingly. It is therefore reasonable to assume that maintained activity spontaneously generated by OKR-related PNC assures this activity balance which is necessary for normal oculomotor function. In order to verify our hypothesis that OKR-related PNC neurons generate spontaneous activity that can be observed in vitro, it will be necessary to identify the postsynaptic targets of the spontaneously active PNC neurons. Conclusions We have been able to demonstrate a specific population of neurons in the PNC that is capable of generating spontaneous activity in vitro. The spontaneous firing depends on a sodium conductance and is independent from afferent synaptic input. Although the postsynaptic target and, consequently, the functional role of the spontaneously active PNC cells remain to be determined it is reasonable to assume that these cells also show spontaneous activity in vivo. Therefore, one likely candidate to represent spontaneously active cells in vivo are PNC neurons that are involved in the generation of slow compensatory eye movements during optokinetic nystagmus. If this is true, spontaneous firing might help to maintain an activity balance between neurons in the right and in the left PNC and thus stabilize eye position in the absence of retinal image motion. Methods Slice preparation Acute brain slices were obtained from 3 to 6 week-old Long-Evans hooded rats of either sex that had been raised at the institute's own colony. All experimental procedures were in strict compliance with governmental regulations and in accordance with the Guidelines for the Use of Animals in Neuroscience Research of the Society for Neuroscience. Animals were deeply anesthetized with halothane and a subcutaneous injection of ketamine (100 mg/kg body weight) and thiazine hydrochloride (1 mg/kg), and transcardially perfused with ice-cold artificial cerebro-spinal fluid (ACSF) containing (in mM), NaCl 123, KCl 2.5, NaH2PO4 1, NaHCO3 26, MgSO4 1.3, CaCl2 1.8, glucose 11, that was continuously gassed with 5% CO2 / 95% O2. After the brain had been removed from the skull, 350 μ m-thick coronal slices were cut on a vibratome in ice cold ACSF. Three to four single slices that included the caudal PNC were obtained from each experimental animal. Slices were kept in ACSF at 36°C for at least one hour to allow recovery from the slicing procedure. For recording, they were transferred to a submerged type recording chamber where they were superfused at 3 ml/min with ACSF at 34°C during patch clamp experiments. Whole-cell patch clamp Whole-cell recordings from neurons in the caudo-lateral PNC were performed under visual guidance using infrared differential interference videomicroscopy [60]. For recording, borosilicate micropipettes (impedance 5–8 MΩ) were filled with internal solution composed of (in mM) potassium gluconate 130, sodium gluconate 5, HEPES 20, MgCl2 4, Na2ATP 4, Na3GTP 0.4, EGTA 0.5, to which 0.5% biocytin was added for morphological single cell reconstruction. Measured membrane potentials were corrected for the junction potential of -10 mV. Postsynaptic responses were evoked with a concentric bipolar stimulation electrode (SNEX-100X, Rhodes Medical Instruments, Tujunga, CA) placed in the optic tract (OT) at the lateral PNC border. Electrical stimuli delivered were 0.5 to 2 mA in amplitude and had a duration of 100 to 500 μs. The neuronal signals were amplified and filtered using an EPC9 amplifier (Heka, Lambrecht, Germany), digitized at 20 kHz, and displayed, stored, and analyzed using PULSE/PULSEFIT software (Heka, Lambrecht, Germany). Unless otherwise stated, postsynaptic current responses evoked by OT stimuli were averaged over three consecutive stimulus applications. All drug effects are given as mean values ± standard deviation, they were statistically tested for significance using the Student's t-test. Drug delivery All drugs used were obtained from Sigma-Aldrich (Deisenhofen, Germany) and were bath applied. A10-minute application time proved sufficient to achieve stable responses. Application of 20 μM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used to block AMPA receptors. Either 50 μM APV or 2 mM kynurenic acid were used to block NMDA receptors. Na currents were suppressed by application of 1 μM tetrodotoxin (TTX). Histochemistry At the end of each recording session, slices were immersion fixed in 4% parafomaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C. After at least 24 h in fixative, slices were processed using standard histochemical techniques for visualization of biocytin with 3,3-diaminobenzidine (Sigma-Aldrich, Deisenhofen, Germany). Morphological reconstruction of stained cells was done with the aid of a camera lucida. Authors' contributions NP participated in the design of the study and executed all aspects including data collection, analysis and drafting the manuscript. MS initially designed the study, assisted with data collection and analysis, and edited the manuscript. Both authors read and approved the final manuscript. Acknowledgements We thank K. Engelhardt for participating in some of the experiments and M. Möllmann for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 509, "Neurovision", TP A8). Figures and Tables Figure 1 Schematic view of stimulation and recording sites. The box in the reconstructed section from the right midbrain in the left panel indicates the position of the PNC shown at higher magnification in the right panel (dorsal is up, medial is to the left). Neurons were recorded from the most dorsal and caudal nuclei of the PNC, the nucleus of the optic tract (NOT) and the posterior pretectal nucleus (PPN). No topographical segregation between spontaneously active cells (indicated by asterisks) and neurons without spontaneous activity (filled circles) was observed within the PNC. The stimulation electrode (black bar) was placed in the optic tract at the dorso-lateral border of the PNC medial to the lateral posterior thalamic nucleus (LP). Other abbreviations used, APN, anterior pretectal nucleus, OPN, olivary pretectal nucleus, LGN, lateral geniculate nucleus, MGB, medial geniculate body, SN, substantia nigra. Figure 2 Different types of responses in PNC neurons evoked by depolarizing current injections. A-C, Whole-cell recordings from three individual PNC cells that showed burst firing in response to depolarizing currents. While the firing rate of cells in A and B monotonically increased with increasing currents, the cell in C only showed spikes in response to intermediate currents. D, Regular non-adapting spiking with monotonically increasing firing rates to increasing currents was observed in another population of cells. E, Responses from a PNC cell that showed irregular fast-adapting spiking. Although increasing currents in this cell decreased interspike intervals the total number of spikes elicited remained constant. Resting potentials of the cells were -61.6 mV, A, -62.5 mV, B, -61.2 mV, C, -58.4 mV, D, and -50.9 mV, E. Current amplitudes applied to each cell were 5, 10, 15, and 20 pA. Figure 3 Morphological and physiological characteristics of PNC neurons with spontaneous activity in vitro. A, B, Reconstruction drawings of two biocytin-filled PNC neurons, insets indicate the cells' position within the PNC. Horizontal lines mark the dorsal border of the PNC, arrowheads in A point to the cell's axon. C, in the absence of injected currents, spontaneous regular spiking was observed in this neuron. D, Responses to intracellular current injection identify this cell as regular non-adapting. E, continuous depolarization starting at a membrane potential of -90 mV revealed this cell's spike threshold at -66.4 mV. d, dorsal, m, medial, v, ventral, l, lateral. Figure 4 Response to intracellular depolarization of spontaneously active PNC cells. Depolarizing current steps induce tonic firing increases in this spontaneously active PNC cell. No phasic component appears in the response to the depolarization step. This behavior was a characteristic property of all spontaneously active PNC cells. Figure 5 Correlation between firing rate and membrane potential in spontaneously active PNC cells. A, Incremental intracellular depolarization leads to increasing firing rates without changing the regular firing pattern. B, When the firing rate is plotted as a function of membrane potential a sinusoidal curve appears that frequently saturates at membrane potentials above -20 mV. Figure 6 Regularity of the firing pattern of spontaneously active PNC cells. A, Current clamp recording from a PNC cell at slightly depolarized membrane potential. B, The interspike interval histogram shows a narrow Gaussian distribution with only little variation of interspike intervals. C, The regularity of the firing can also be derived from the autocorrelogram which exhibits multiple equally spaced peaks due to the little variation in the interspike intervals. Figure 7 Spontaneous activity of PNC is independent from tonic excitatory input. A single electric shock delivered to the optic tract lateral from the recorded neuron evokes a single peak EPSC (A) that completely disappears after AMPA receptor blockade by bath application of 20 μM CNQX (E). In contrast, the spontaneous firing rate recorded in control situation (B) does not change after AMPA receptor blockade (F). Removing excitatory input does also not change the regularity of the firing as derived from the distribution of interspike intervals (C vs. G) or from the autocorrelogram (D vs. H). Figure 8 Tonic synaptic input does not contribute to the generation of spontaneous activity in PNC cells. Postsynaptic responses obtained by electrical optic tract stimulation (A) can also be blocked by bath application of 1.5 mM CoCl2 (D). However, the spontaneous firing rate recorded in control situation (B) does not change after removal of synaptic input (E). Removing excitatory input does also not change the regularity of the firing as derived from the distribution of interspike intervals (C vs. F). Figure 9 Spontaneous activity in PNC cells depends on a sodium conductance. In control solution (A) this cell showed regular firing pattern with depolarizing inward currents preceding each action potential. When 1 μM TTX was added to the bath the firing rate dropped at the beginning of TTX application (B). In the presence of TTX, both action potentials and depolarizing inward currents completely disappeared 5 min later (C). ==== Refs Distler C Hoffmann KP The pupillary light reflex in normal and innate microstrabismic cats, I: Behavior and receptive-field analysis in the nucleus praetectalis olivaris Vis Neurosci 1989 3 127 138 2487096 Distler C Hoffmann KP The pupillary light reflex in normal and innate microstrabismic cats, II: Retinal and cortical input to the nucleus praetectalis olivaris Vis Neurosci 1989 3 139 153 2487097 Gamlin PD Zhang H Clarke RJ Luminance neurons in the pretectal olivary nucleus mediate the pupillary light reflex in the rhesus monkey Exp Brain Res 1995 106 169 176 8542972 Klooster J Vrensen GF Muller LJ Van der Want JJ Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes. A light microscopic tracing study Brain Res 1995 688 34 46 8542320 10.1016/0006-8993(95)00497-E Kourouyan HD Horton JC Transneuronal retinal input to the primate Edinger-Westphal nucleus J Comp Neurol 1997 381 68 80 9087420 10.1002/(SICI)1096-9861(19970428)381:1<68::AID-CNE6>3.0.CO;2-I Pong M Fuchs AF Characteristics of the pupillary light reflex in the macaque monkey: discharge patterns of pretectal neurons J Neurophysiol 2000 84 964 974 10938320 Clarke RJ Zhang H Gamlin PD Primate pupillary light reflex: receptive field characteristics of pretectal luminance neurons J Neurophysiol 2003 89 3168 3178 12611972 Collewijn H Direction-selective units in the rabbit's nucleus of the optic tract Brain Res 1975 100 489 508 172194 10.1016/0006-8993(75)90154-7 Hoffmann KP Behrend K Schoppmann A A direct afferent visual pathway from the nucleus of the optic tract to the inferior olive in the cat Brain Res 1976 115 150 153 974739 10.1016/0006-8993(76)90830-1 Kato I Harada K Hasegawa T Igarashi T Koike Y Kawasaki T Role of the nucleus of the optic tract in monkeys in relation to optokinetic nystagmus Brain Res 1986 364 12 22 3947958 10.1016/0006-8993(86)90982-0 Schiff D Cohen B Büttner-Ennever J Matsuo V Effects of lesions of the nucleus of the optic tract on optokinetic nystagmus and after-nystagmus in the monkey Exp Brain Res 1990 79 225 239 2323371 Cohen B Reisine H Yokota JI Raphan T The nucleus of the optic tract. Its function in gaze stabilization and control of visual-vestibular interaction Ann NY Acad Sci 1992 656 277 296 1599149 Fuchs AF Mustari MJ Robinson FR Kaneko CR Visual signals in the nucleus of the optic tract and their brain stem destinations Ann NY Acad Sci 1992 656 266 276 1599148 Schmidt M Hoffmann KP Physiological identification of pretectal neurons projecting to the lateral geniculate nucleus in the cat Eur J Neurosci 1992 4 318 326 12106358 Schmidt M Neurons in the cat pretectum that project to the dorsal lateral geniculate nucleus are activated during saccades J Neurophysiol 1996 76 2907 2918 8930243 Fischer WH Schmidt M Hoffmann KP Saccade-induced activity of dorsal lateral geniculate nucleus X- and Y- cells during pharmacological inactivation of the cat pretectum Vis Neurosci 1998 15 197 210 9605522 10.1017/S0952523898151106 Schmidt M Sudkamp S Wahle P Characterization of pretectal-nuclear-complex afferents to the pulvinar in the cat Exp Brain Res 2001 138 509 519 11465750 10.1007/s002210100738 Terasawa K Otani K Yamada J Descending pathways of the nucleus of the optic tract in the rat Brain Res 1979 173 405 417 385106 10.1016/0006-8993(79)90238-5 Lugo-Garcia N Kicliter E Superior colliculus efferents to five subcortical visual system structures in the ground squirrel Brain Res 1987 426 131 141 3690310 10.1016/0006-8993(87)90432-X Reber A Sarrau JM Carnet J Magnin M Stelz T Horizontal optokinetic nystagmus in unilaterally enucleated pigmented rats: role of the pretectal commissural fibers J Comp Neurol 1991 313 604 612 1783684 Vargas CD Volchan E Hokoc JN Pereira A Bernardes RF Rocha-Miranda CE On the functional anatomy of the nucleus of the optic tract-dorsal terminal nucleus commissural connection in the opossum (Didelphis marsupialis aurita) Neuroscience 1997 76 313 321 8971781 10.1016/S0306-4522(96)00356-9 Watanabe S Kato I Hattori K Azuma M Nakamura T Koizuka I Effects on the optokinetic system of midline lesions in the pretectum of monkeys J Vestib Res 2001 11 73 80 11847451 Baldauf ZB Wang XP Wang S Bickford ME Pretectotectal pathway: an ultrastructural quantitative analysis in cats J Comp Neurol 2003 464 141 158 12898608 10.1002/cne.10792 Schmidt M Schiff D Bentivoglio M Independent efferent populations in the nucleus of the optic tract: an anatomical and physiological study in rat and cat J Comp Neurol 1995 360 271 285 8522647 Sudkamp S Schmidt M Physiological characterization of pretectal neurons projecting to the lateral posterior-pulvinar complex in the cat Eur J Neurosci 1995 7 881 888 7613624 Young MJ Lund RD The retinal ganglion cells that drive the pupilloconstrictor response in rats Brain Res 1998 787 191 202 9518608 10.1016/S0006-8993(97)01473-X Hoffmann KP Distler C Erickson RG Mader W Physiological and anatomical identification of the nucleus of the optic tract and dorsal terminal nucleus of the accessory optic tract in monkeys Exp Brain Res 1988 69 635 644 3371443 Volchan E Rocha-Miranda CE Picanco-Diniz CW Zinsmeisser B Bernardes RF Franca JG Visual response properties of pretectal units in the nucleus of the optic tract of the opossum Exp Brain Res 1989 78 380 386 2599047 10.1007/BF00228910 Hoffmann KP Distler C Quantitative analysis of visual receptive fields of neurons in nucleus of the optic tract and dorsal terminal nucleus of the accessory optic tract in macaque monkey J Neurophysiol 1989 62 416 428 2769338 Klauer S Sengpiel F Hoffmann KP Visual response properties and afferents of nucleus of the optic tract in the ferret Exp Brain Res 1990 83 178 189 2073937 10.1007/BF00232207 Mustari MJ Fuchs AF Discharge patterns of neurons in the pretectal nucleus of the optic tract (NOT) in the behaving primate J Neurophysiol 1990 64 77 90 2388076 Ibbotson MR Mark RF Maddess TL Spatiotemporal response properties of direction-selective neurons in the nucleus of the optic tract and dorsal terminal nucleus of the wallaby, Macropus eugenii J Neurophysiol 1994 72 2927 2943 7897500 Ilg UJ Hoffmann KP Responses of neurons of the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic tract in the awake monkey Eur J Neurosci 1996 8 92 105 8713453 Schweigart G Hoffmann KP Pretectal jerk neuron activity during saccadic eye movements and visual stimulations in the cat Exp Brain Res 1992 91 273 283 1459229 10.1007/BF00231660 Ibbotson MR Mark RF Wide-field nondirectional visual units in the pretectum: do they suppress ocular following of saccade-induced visual stimulation J Neurophysiol 1994 72 1448 1450 7807228 Price NS Ibbotson MR Pretectal neurons optimized for the detection of saccade-like movements of the visual image J Neurophysiol 2001 85 1512 1521 11287475 Inouye S Kawamura H Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus Proc Natl Acad Sci USA 1979 76 5962 5966 293695 Groos G Hendriks J Circadian rhythms in electrical discharge of rat suprachiasmatic neurones recorded in vitro Neurosci Lett 1982 34 283 288 6298675 10.1016/0304-3940(82)90189-6 Bos NPA Mirmiran M Circadian rhythms in spontaneous neuronal discharges of the cultured suprachiasmatic nucleus Brain Res 1990 511 158 162 2331613 10.1016/0006-8993(90)90235-4 Bennett BD Wilson CJ Spontaneous activity of neostriatal cholinergic interneurons in vitro J Neurosci 1999 19 5586 5596 10377365 Sanghera MK Trulson ME German DC Electrophysiological properties of mouse dopamine neurons: in vivo and in vitro studies Neuroscience 1984 12 793 801 6472621 10.1016/0306-4522(84)90171-4 Grace AA Onn S-P Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro J Neurosci 1989 9 3463 3481 2795134 Nakanishi H Kita H Kitai ST Electrical membrane properties of rat subthalamic neurons in an in vitro slice preparation Brain Res 1987 437 35 44 3427481 10.1016/0006-8993(87)91524-1 Bevan MD Wilson CJ Mechanisms underlying spontaneous oscillation and rhythmic firing in rat subthalamic neurons J Neurosci 1999 19 7617 7628 10460267 Ujihara H Akaike A Sasa M Takaori S Electrophysiological evidence for cholinoceptive neurons in the medial vestibular nucleus: studies on rat brain stem in vitro Neurosci Lett 1988 93 231 235 3241648 10.1016/0304-3940(88)90087-0 Darlington CL Smith PF Hubbard JI Neuronal activity in the guinea pig medial vestibular nucleus in vitro following chronic unilateral labyrinthectomy Neurosci Lett 1989 105 143 148 2485877 10.1016/0304-3940(89)90026-8 Dutia MB Johnston AR McQueen DS Tonic activity of rat medial vestibular nucleus neurones in vitro and its inhibition by GABA Exp Brain Res 1992 88 466 472 1587312 Häusser M Clark BA Tonic synaptic inhibition modulates neuronal output pattern and spatiotemporal synaptic integration Neuron 1997 19 665 678 9331356 10.1016/S0896-6273(00)80379-7 Jahnsen H Electrophysiological characteristics of neurones in the guinea-pig deep cerebellar nuclei in vitro J Physiol (Lond) 1986 372 129 147 3723407 Hounsgaard J Pacemaker properties of mammalian Purkinje cells Acta Physiol Scand 1979 106 91 92 463582 Llinás R Sugimori M Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices J Physiol (Lond) 1980 305 197 213 7441553 McCormick DA Pape H-C Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay cells J Physiol (Lond) 1990 431 291 318 1712843 Feigenspan A Gustincich S Bean SP Raviola E Spontaneous activity of solitary dopaminergic cells of the retina J Neurosci 1998 18 6776 6789 9712649 Pennartz CMA Bierlaagh MA Geurtsen AMS Cellular mechanisms underlying spontaneous firing in rat suprachiasmatic nucleus: involvement of a slowly inactivating component of sodium current J Neurophysiol 1997 78 1811 1825 9325350 Zhang H Clarke RJ Gamlin PD Behavior of luminance neurons in the pretectal olivary nucleus during the pupillary near response Exp Brain Res 1996 112 158 162 8951418 Simpson JI Giolli RA Blanks RHI Büttner-Ennever J The pretectal nuclear complex Neuroanatomy of the oculomotor system 1988 Amsterdam, Elsevier 335 364 Missal M Coimbra A Lefevre P Olivier E A quantitative analysis of the correlations between eye movements and neural activity in the pretectum Exp Brain Res 2002 143 373 382 11889515 10.1007/s00221-002-0999-7 Hoffmann KP Stone J Retinal input to the nucleus of the optic tract of the cat assessed by antidromic activation of ganglion cells Exp Brain Res 1985 59 395 403 4029313 10.1007/BF00230920 Hoffmann KP Fischer WH Directional effect of inactivation of the nucleus of the optic tract on optokinetic nystagmus in the cat Vision Res 2001 41 3389 3398 11718781 10.1016/S0042-6989(01)00184-5 Dodt HU Zieglgänsberger W Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy Brain Res 1990 537 333 336 2085783 10.1016/0006-8993(90)90380-T

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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-171532915410.1186/1471-2210-4-17Research ArticleThe aminoguanidine carboxylate BVT.12777 activates ATP-sensitive K+ channels in the rat insulinoma cell line, CRI-G1 Kinsella Jackie M [email protected] Hilary A [email protected] Teresa [email protected] Jenni [email protected] Calum [email protected] Michael LJ [email protected] Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK2004 24 8 2004 4 17 17 22 4 2004 24 8 2004 Copyright © 2004 Kinsella et al; licensee BioMed Central Ltd.2004Kinsella et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background 3-guanidinopropionic acid derivatives reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, the aminoguanidine carboxylate BVT.12777, opens KATP channels in rat insulinoma cells, by the same mechanism as leptin. Results BVT.12777 hyperpolarized CRI-G1 rat insulinoma cells by activation of KATP channels. In contrast, BVT.12777 did not activate heterologously expressed pancreatic β-cell KATP subunits directly. Although BVT.12777 stimulated phosphorylation of MAPK and STAT3, there was no effect on enzymes downstream of PI3K. Activation of KATP in CRI-G1 cells by BVT.12777 was not dependent on MAPK or PI3K activity. Confocal imaging showed that BVT.12777 induced a re-organization of cellular actin. Furthermore, the activation of KATP by BVT.12777 in CRI-G1 cells was demonstrated to be dependent on actin cytoskeletal dynamics, similar to that observed for leptin. Conclusions This study shows that BVT.12777, like leptin, activates KATP channels in insulinoma cells. Unlike leptin, BVT.12777 activates KATP channels in a PI3K-independent manner, but, like leptin, channel activation is dependent on actin cytoskeleton remodelling. Thus, BVT.12777 appears to act as a leptin mimetic, at least with respect to KATP channel activation, and may bypass up-stream signalling components of the leptin pathway. ==== Body Background ATP-sensitive K+ (KATP) channels are important regulators of cell function, coupling energy metabolism with electrical activity. KATP channels are comprised of two proteins, derived from the sulphonylurea receptor (SUR) family and an inwardly rectifying K+ channel (Kir6.x family), the exact composition of these being dependent upon tissue [1,2]. For example, pancreatic β-cells and insulin-secreting clonal cell lines express KATP channels consisting of Kir6.2 and SUR1 subunits [3]. KATP channels are present in numerous tissues and are the target for drugs that inhibit or increase channel activity [4,5]. The archetypal inhibitors of these channels are the sulphonylurea class of drugs, which bind to the SUR subunit of the channel. Modulation of KATP channel activity in pancreatic β-cells has profound effects on insulin secretion and glucose homeostasis [6]. Sulphonylureas such as tolbutamide and glibenclamide inhibit channel activity, resulting in β-cell depolarization, increased electrical activity, enhanced calcium entry and consequently increased insulin secretion [7]. In contrast, pancreatic β-cell KATP channel activation induces hyperglycaemia in animals and man [8]. This latter action is caused by membrane hyperpolarization, reduction in cell excitability and decreased intracellular calcium resulting in reduced secretion of insulin. Such effects have been reported following application of the benzothiadiazine, diazoxide, which has been used on occasion to treat persistent hyperinsulinemic hypoglycaemia of infancy [8]. It has been demonstrated that diazoxide interacts with the sulphonylurea receptor subunit, SUR1, encompassing transmembrane domains 6–11 and the first nucleotide binding fold [9]. A similar conclusion has also been reached using a novel diazoxide analogue [10]. The presence of KATP channels in many other tissues, notably muscle and central neurons, has stimulated interest in the development of novel, selective KATP channel openers for the treatment of various diseases [10,11]. The ob gene product leptin has been demonstrated to activate KATP channels in pancreatic β-cells [12] and insulin-secreting cell lines [13], consistent with a potential role in modifying insulin secretion [14]. One of the primary functions for this hormone is its role in the regulation of food intake and body weight [15]. Interestingly, leptin also activates KATP channels of hypothalamic glucose-responsive neurones [16,17] indicating a possible role for this channel in the control of energy homeostasis and body weight. In addition, Kir6.2 knock-out mice have deficits in central glucose sensing leading to loss of glucose mediated feeding response and a defective hypoglycaemic compensatory response [18]. These latter findings suggest that hypothalamic KATP channels may also be an important target for drug manipulation with respect to centrally driven control of glucose and energy homeostasis. The aminoguanidine carboxylate, BVT.12777 (Figure 1), is one of a series of structurally related molecules based on the anti-diabetic/anti-obesity agent 3-guanidinopropionic acid [19], which, like leptin, have been demonstrated to reduce body weight in obese diabetic (ob/ob) mice [20]. Here we demonstrate that BVT.12777 opens KATP channels in the CRI-G1 insulin secreting cell line, a useful model for pancreatic β-cells [21], and for analysing the mechanism by which leptin opens KATP channels [13,22,23]. Figure 1 Structure of BVT.12777 ([2-(hydrazinoiminomethyl)-hydrazino] acetic acid) Results BVT.12777 activates KATP channels Under current clamp conditions with 5 mM ATP in the pipette solution to maintain KATP channels in the closed state, the mean resting potential was -38.7 ± 1.7 mV (n = 10), similar to values reported in previous studies [13,22] under these recording conditions. Application of BVT.12777 (100 μM) hyperpolarized CRI-G1 cells (Figure 2A) to -66.3 ± 2.7 mV (n = 10). Examination of the voltage-clamped macroscopic currents indicates that prior to the addition of BVT.12777 the slope conductance of the cells was 0.43 ± 0.03 nS (n = 10), and following exposure to BVT.12777 (100 μM), this increased to 3.45 ± 1.17 nS (n = 10). The reversal potential (obtained from the point of intersection of the current-voltage relationship) associated with the BVT.12777-induced conductance increase (Figure 2A) was -78.5 ± 0.8 mV (n = 10), close to the calculated value for Ek of -84 mV in this system, indicating increased K+ conductance. CRI-G1 cells responded to BVT.12777 in an all or none manner, with cells undergoing full hyperpolarization and increase in conductance, at all concentrations (100 – 300 μM) examined. Such an effect has also been reported for leptin on CRI-G1 cells [13]. Removal of BVT.12777 from the bath solution did not fully recover the membrane potential and conductance to control values over the next 15–30 minutes (not shown). Application of the KATP channel inhibitor, tolbutamide (100 μM) during BVT.12777 exposure (Figure 2A) completely reversed the BVT.12777-induced hyperpolarization and decreased conductance, to -41.0 ± 4.8 mV (n = 5) and 0.58 ± 0.07 nS (n = 5) respectively, values indistinguishable from control (P > 0.05). These data indicate that BVT.12777 increases KATP current in this cell line. This is demonstrated more clearly in cell-attached recordings from CRI-G1 cells, where bath application of BVT.12777 (100 μM) resulted in activation of single KATP channel currents (Figure 2B; n = 7). The increase in channel activity was evident within 5 minutes of drug application, was sustained over the time course of exposure (~30 minutes) and was not immediately reversed following removal of the drug. Figure 2C shows mean channel activity (Nf.Po), normalised to the control for each recording, plotted against time of exposure to BVT.12777. BVT.12777 activation of KATP channels was demonstrated to be reversibly inhibited by 100 μM tolbutamide (n = 4; Figure 2B,2C). Identical control experiments, in the absence of BVT.12777, resulted in no significant effect on KATP channel activity, over a 30-minute test period (n = 8; P > 0.05). Figure 2 BVT.12777 activates a tolbutamide-sensitive K+ current A, the upper trace shows a current clamp recording of a CRI-G1 cell following dialysis with a 5 mM ATP-containing solution. In this and subsequent current clamp figures the trace begins approximately 5 min after formation of the whole-cell configuration. Application of BVT.12777 (100 μM) for the time indicated hyperpolarized the cell from -50 mV to -76 mV, an action readily reversed by tolbutamide (100 μM), which returned membrane potential to -54 mV. Washout of all drugs from the bath resulted in a membrane potential of -70 mV, indicating the lack of reversibility of BVT.12777. The lower plot is the current-voltage relationship for the voltage clamped currents. Cells were voltage clamped at -50 mV and 10 mV steps of 100 ms duration were applied every 200 ms (range -120 to -30 mV). BVT.12777 increased the membrane conductance relative to control and tolbutamide reversed this BVT.12777-induced conductance increase with a reversal potential of -78 mV. B, cell-attached recording from a CRI-G1 cell, at 10 mV applied to the recording pipette. Single channel openings are shown as downward deflections. Addition of 100 μM BVT.12777 induced an increase in channel activity (Nf.Po) from 0.17 in control to 0.31, and 1.25 at 10 and 20 minutes respectively, after BVT addition. Application of 100 μM tolbutamide induced a substantial inhibition of activity (to 0.02), which was reversed on washout of all drugs, with activity increasing to 0.74. The symbol C refers to the closed state of the channel in this and subsequent figures. C, diary plot of Nf.Po against time from cell-attached experiments in the presence and absence of BVT.12777, where channel activity was calculated every 2 minutes. Each point is the mean of 4–7 separate determinations. The effect of BVT.12777 on KATP channel activity in excised membrane patches was also examined. Recordings were made from inside-out patches in symmetrical (140 mM KCl in pipette and bath solutions) K+ at a membrane potential of -40 mV. KATP channels were identified by inhibition of channel activity following application of 100 μM MgATP to the inner membrane aspect of the patch, which reduced normalised NfPo from 1.0 to 0.23 ± 0.05 (n = 4; P < 0.05). Subsequent application of 100 μM BVT.12777, in the continued presence of MgATP, induced a gradual increase in KATP channel activity (Figure 3), to levels similar to that of control (in the absence of MgATP). For example 15 minutes after 100 μM BVT.12777 application normalised mean channel activity had recovered to 1.18 ± 0.46 (n = 4). In experiments where no drug was added, KATP channel currents, in the presence of 100 μM MgATP, did not activate spontaneously (n = 4). Figure 3 BVT.12777 activates KATP channels in inside-out patches Continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited channel activity by >90%, demonstrating KATP identity. Addition of 100 μM BVT.12777, in the presence of 100 μM MgATP to the cytoplasmic aspect of the patch resulted in KATP channel activation. Nf.Po values were 2.96 (control, after first MgATP challenge), and 0.25 in the presence of MgATP, which increased to 0.72, 1.06 and 2.74 at 5, 10 and 20 minutes respectively, after BVT.12777 addition. BVT.12777 activates KATP channels independently of PI 3-kinase activity Leptin and diazoxide hyperpolarized CRI-G1 cells, in a manner similar to that of BVT.12777 (data not shown). Leptin (10 nM) induced a hyperpolarization from a mean membrane potential of -47.6 ± 1.6 mV to -68.5 ± 1.9 mV (n = 8; P < 0.05), and application of tolbutamide (100 μM) reversed this action, returning the membrane potential to -47.5 ± 1.9 mV (n = 4). Diazoxide (200 μM) rapidly hyperpolarized CRI-G1 cells from a mean membrane potential of -49.9 ± 1.7 mV to -74.0 ± 1.5 mV (n = 6; P < 0.05), with tolbutamide (100 μM) also reversing this action, returning membrane potential to -46.9 ± 3.8 mV (n = 6). Leptin, but not diazoxide activation of CRI-G1 KATP channels is PI3K dependent [22,23]. Thus, we investigated whether BVT.12777 activates KATP channels in CRI-G1 cells by direct (like diazoxide) or indirect (like leptin) mechanisms. Pre-incubation of CRI-G1 cells (20 min) with inhibitors of PI 3-kinase, wortmannin (10 nM) or LY294002 (10 μM) had no significant effect on the mean resting membrane potential or slope conductance of CRI-G1 cells and did not prevent BVT.12777 from causing hyperpolarization and increased cell conductance (Figure 4A). In the presence of 10 nM wortmannin, values for mean membrane potential and slope conductance were -44.3 ± 1.2 mV (n = 6) and 0.86 ± 0.10 nS (n = 5), and addition of 200 μM BVT.12777 hyperpolarized cells to -68.9 ± 0.8 mV (n = 6) with an increase in slope conductance to 3.10 ± 0.38 nS (n = 5). Identical results were obtained in the presence of 10 μM LY294002 (data not shown), with corresponding control values of -40.8 ± 2.8 mV (n = 6) and 0.79 ± 0.11 nS (n = 4), and in the presence of 200 μM BVT.12777, -67.9 ± 0.6 mV (n = 6) and 2.69 ± 0.35 nS (n = 4) for membrane potential and slope conductance respectively. In all experiments (i.e with either PI3K inhibitor) addition of tolbutamide (100 μM) recovered the membrane potential (-41.8 ± 1.5 and -34.0 ± 1.7 mV; n = 6) and slope conductance (0.89 ± 0.11 (n = 5) and 0.58 ± 0.06 (n = 4) nS) for wortmannin and LY294002 respectively, to values indistinguishable from controls (P > 0.1). Cell-attached recordings from CRI-G1 cells also show that wortmannin (10 – 100 nM) did not occlude BVT.12777 activation of KATP channels (Figure 4B). Mean channel activity in the presence of wortmannin (10 nM) was 0.02 ± 0.00 which increased to 0.16 ± 0.02, 20 minutes after exposure to 100 μM BVT.12777 (n = 3; P < 0.05). Control experiments where no BVT.12777 was added show no change in channel activity over a 30-minute period (Nf.Po = 0.01 ± 0.00 and 0.04 ± 0.00 after 5 and 30 minutes respectively; n = 4). Figure 4 Wortmannin does not inhibit BVT.12777 activation of KATP A, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Application of BVT.12777 (200 μM), in the continued presence of wortmannin hyperpolarized the cell from -46 to -77 mV. Tolbutamide (100 μM), applied after the BVT-induced hyperpolarization, recovered the membrane potential (to -40 mV). B, cell-attached recordings from CRI-G1 cells, following exposure of cells to 10 nM wortmannin for 15–20 minutes. Upper trace; in the continued presence of wortmannin, Nf.Po was 0.01 and 0.03 after 5 and 30 minutes respectively. Lower trace, application of BVT.12777 (100 μM) to cell-attached recording in the presence of 10 nM wortmannin resulted in KATP activation, with Nf.Po values of 0.01, 0.12 and 0.27 prior to, and 10 and 30 minutes after, BVT.12777, respectively. Addition of 100 nM wortmannin did not inhibit channel activity. Heterologously expressed KATP currents are not activated by BVT.12777 Oocytes injected with Kir6.2 and SUR1 cRNAs were challenged with sodium azide (3 mM) to elicit a reversible increase in current, which was completely blocked by 1 μM glibenclamide or 0.5 mM tolbutamide, indicating that the current was due to KATP activation, as described previously [24,25]. In oocytes, previously exposed to sodium azide in order to verify Kir6.2-SUR1 expression, application of BVT.12777 (10 μM – 1 mM) did not produce any consistent increase in KATP current (n = 16; data not shown). Consequently, we utilized an alternative expression system, the HEK 293 cell line [25]. Application of BVT.12777 (100 μM) to the bathing solution using the cell-attached recording configuration resulted in no significant increase in mean channel activity above control levels over a 30-minute period, although subsequent addition of sodium azide (3 mM) did cause a rapid increase in channel activity, which was reversed by the addition of 100 μM tolbutamide (n = 4, data not shown). Similarly, application of BVT.12777 in the presence of 0.1 mM MgATP to inside-out patches from HEK 293 cells transiently expressing Kir6.2-SUR1, did not cause activation of channel activity following 30 minutes exposure (n = 4; data not shown). Thus BVT.12777 does not appear to be capable of activating heterologously expressed Kir6.2-SUR1 currents. MAPK does not mediate BVT.12777-activation of KATP Exposure of CRI-G1 cells to BVT.12777 (100 μM) for up to 30 minutes had no consistent effect on the phosphorylation of enzymes downstream of PI3K (PKB and its downstream target, GSK3), but did increase the phosphorylation of STAT3 (n = 4) and MAPK (n = 4; data not shown). These data are in agreement with the lack of BVT.12777 sensitivity to PI3K inhibitors on activation of KATP channels. However, activation of MAPK has been implicated as a significant intermediate for both insulin and leptin signalling pathways in various cell types [26-29]. Thus, we examined the effect of UO126, a potent and specific inhibitor of the activation of the classical MAPK cascade [30], on BVT.12777 opening of KATP channels. Application of UO126 (25 μM) inhibited approximately 90 % of KATP channel activity in cell-attached or inside-out recordings, whereas 1–10 μM UO126, concentrations that suppresses activation of MAPKK [30], had no significant effect on channel activity (data not shown). Control cell attached recordings had a mean channel activity of 0.07 ± 0.02, which increased to 1.29 ± 0.82 (n = 3) in the presence of BVT.12777 (100 μM). Subsequent application of UO126 (1 μM) in the continued presence of BVT.12777 did not alter channel activity (data not shown), over a 15-minute period (Nf.Po was 1.86 ± 1.45 (n = 3) and 2.44 ± 2.00 (n = 3), at 5 and 15 minutes respectively; P < 0.05). In addition, increasing UO126 to 10 μM had no effect on BVT.12777 induced KATP channel activation. BVT.12777 activation of KATP channels is dependent on actin cytoskeleton dynamics Leptin activation of KATP channels in the CRI-G1 cell line is dependent upon reorganisation of the cytoskeleton, a process downstream from PI3K activation [31]. Therefore, we examined whether BVT.12777 opening of CRI-G1 KATP channels occurs through alteration of actin filament dynamics. For this series of experiments the heptapeptide mushroom toxin phalloidin [32] was used to stabilise the polymerised form of actin (F-actin). As phalloidin is membrane-impermeant, it was directly applied to the internal aspect of the cell membrane. In whole-cell experiments, 10 μM phalloidin was added to the electrode solution and allowed to dialyse into the cell. The mean resting potential and slope conductance were -38.0 ± 0.6 mV and 0.66 ± 0.04 nS (n = 4) respectively, and following addition of 200 μM BVT.12777 no significant change in these parameters was observed (Figure 5A), with a mean membrane potential of -41.7 ± 1.1 mV and slope conductance of 0.60 ± 0.08 nS (n = 4; P > 0.05). The presence of phalloidin (10 μM) in the bath solution also prevented KATP channel activation by BVT.12777 in the inside-out isolated patch configuration (Figure 5B). Application of 0.1 mM MgATP to the cytoplasmic aspect of inside-out patches caused 97.5 ± 2.1% inhibition of KATP channel activity (n = 3; P < 0.05) and subsequent addition of 10 μM phalloidin had no further effect, as reported previously [29]. Subsequent addition of BVT.12777 (100 μM) failed to increase KATP channel activity, with mean Nf.Po values of 0.06 ± 0.05 and 0.03 ± 0.01 in the absence and presence of BVT.12777 respectively (n = 3; P > 0.05). In contrast, the direct KATP channel opener, diazoxide activates KATP channels in the presence of phalloidin. In whole-cell experiments (Figure 5C), diazoxide (200 μM) hyperpolarized CRI-G1 cells from a mean membrane potential of -42.6 ± 0.1 mV to -70.1 ± 0.8 mV (n = 4; P < 0.05), and increased slope conductance from 0.87 ± 0.23 to 7.39 ± 0.72, actions reversed by tolbutamide (100 μM). Figure 5 Phalloidin prevents BVT.12777 activation of KATP A, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of BVT.12777 (200 μM) had no effect on the membrane potential of the cell (-41 mV) in the presence of phalloidin. B, continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited Nf.Po from 1.25 to 0.02. Addition of 10 μM phalloidin and subsequently 100 μM BVT.12777, in the presence of 100 μM MgATP, to the cytoplasmic aspect of the patch resulted in no effect on KATP, with Nf.Po values of 0.01 and 0.03 respectively. C, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of diazoxide (200 μM) induced rapid cell membrane hyperpolarization, from -55 to -72 mV, an action reversed (to -45 mV) by tolbutamide (100 μM). F-actin is disrupted by BVT.12777 The prevention of BVT.12777-induced KATP activation by phalloidin mirrors the effect of this toxin on leptin activation of KATP [31]. Thus, we visualised F-actin by staining with rhodamine-conjugated phalloidin. In untreated CRI-G1 cells there was pronounced phalloidin-positive labelling of the cell membrane, with more diffuse, granular staining within the cytoplasm (Figure 6A). In contrast, cells treated with BVT.12777 (100 μM) or leptin (10 nM) for 40 min showed a marked reduction in phalloidin fluorescence intensity, with disjointed labelling at the cell membrane (Figure 6A). The actin filament disrupter cytochalasin B [33] also reduced the intensity of phalloidin labelling but in a more punctate manner on visualisation of treated cells compared with controls (data not shown). Analysis of the mean fluorescence intensity at the cell membrane following the actions of BVT.12777 and leptin demonstrated that both treatments caused a significant reduction of the intensity of rhodamine-phalloidin labelling, by 43.0 ± 4.2% (n = 6; P < 0.05) and 62.2 ± 6.0% (n = 6; P < 0.05), respectively, compared to untreated cells (Figure 6B). However, the directly acting KATP channel opener, diazoxide did not cause disruption of the actin cytoskeleton (Figure 6A,6B), with a relative intensity of rhodamine-phalloidin staining of 0.98 ± 0.16 (P > 0.05). Figure 6 BVT.12777 disrupts the actin cytoskeleton A, images of rhodamine-conjugated phalloidin fluorescence in CRI-G1 cells in control conditions and following incubation with leptin (10 nM), BVT.12777 (100 μM) or diazoxide (200 μM) for 30 minutes. All panels show representative X-Y images. Note the marked reduction in phalloidin staining in cells pre-treated with leptin or BVT.12777, and not diazoxide. Scale bars are 50 μm. B, histogram comparing the normalised fluorescence intensity relative to control in the membrane periphery of randomly selected CRI-G1 cells for each condition; (control (n = 13; cells = 195), 10 nM leptin (n = 6; cells = 90), BVT.12777 (n = 6; cells = 90) and 200 μM diazoxide (n = 4, cells = 60). Error bars indicate s.e.m. and * significance of P < 0.001. Discussion BVT.12777 induced hyperpolarization of CRI-G1 cells, with an associated increase in K+ conductance, an action likely caused by the activation of KATP channels, as the sulphonylurea tolbutamide completely reversed its effects. Cell-attached and inside-out single channel current recordings demonstrate directly that BVT.12777 activates KATP channels. The increased KATP current generated in isolated membrane patches resembles the effects of KATP activators such as diazoxide [34] and sodium azide [35], which have also been shown to activate insulinoma or pancreatic β-cell KATP channels in isolated patches in the presence of Mg-ATP. Thus, although not tested here, BVT.12777 as an activator of KATP would be expected, as observed for diazoxide, to inhibit insulin release from CRI-G1 cells stimulated by metabolizable substrates or tolbutamide [36], although this would clearly be dependent on its action on other β-cell conductances, notably calcium channels. BVT.12777 activation of KATP channels was only slowly reversed on withdrawal of the drug, unlike the actions of diazoxide or sodium azide, which are rapidly reversed on washout [35,36]. Indeed, following removal of BVT.12777 in the absence or presence of tolbutamide, enhanced KATP channel activity was apparent for a considerable time. The slow reversibility on washout of BVT.12777 resembles the effects of the hormone leptin on CRI-G1 cell membrane potential and KATP channel activation [13]. Leptin, via activation of the main signalling form of the leptin receptor (ObRb), has been shown to increase the phosphorylation of STAT3, MAPK and to stimulate PI3K pathways in various peripheral tissues, cell lines [37], and in hypothalamic neurones [38]. BVT.12777 although stimulating phosphorylation of STAT3 and MAPK did not stimulate PI3K dependent pathways as demonstrated by the lack of effect on the phosphorylation status of the PI3K output indicators, PKB and GSK3. It is unclear at present how this molecule induces STAT3 and MAPK phosphorylation. As KATP activation by BVT.12777 is rapid and occurs in isolated membrane patches it is unlikely that any JAK-STAT pathway (which drives changes in transcription) contributes to this action. Leptin activation of KATP channel currents in CRI-G1 cells has previously been shown to be independent of MAPK, but prevented by the inhibitors of PI3K [22]. However, BVT.12777 activation was not only insensitive to inhibition by the MAPKK inhibitor, UO126, it was also insensitive to the presence of the PI3-kinase inhibitors, wortmannin and LY294002, at concentrations sufficient to prevent leptin activation of KATP in this cell line. These data led us to suspect that BVT.12777, irrespective of its ability to initiate various signalling cascades in this cell line, increased KATP channel activity by a more direct effect on the channel subunits in a manner analogous to diazoxide, which is purported to interact directly with the SUR1 subunit [9,10]. This possibility was tested by heterologous expression of the β-cell subunits of KATP channels, Kir6.2 and SUR1, in Xenopus oocytes, a commonly utilised expression system for electrophysiological studies of these recombinant channels [24,25]. However, BVT.12777 did not activate Kir6.2-SUR1 currents in oocytes, demonstrated to express functional KATP channel currents. Thus we explored this question further by utilising a second heterologous expression system for Kir6.2-SUR1, HEK293 cells. Recordings from inside-out patches demonstrated that BVT.12777 did not activate Kir6.2-SUR1 currents in the presence of Mg-ATP, in contrast to diazoxide [39] or sodium azide [35]. Overall these data strongly suggest that expression of the KATP channel subunits, Kir6.2 and SUR1 are insufficient per se to bring about sensitivity to BVT.12777, and indicate that this opener may activate this channel type by an indirect mechanism (which is not available in oocytes or HEK cells). Although the activation of KATP channels by leptin in CRI-G1 cells is PI3-kinase dependent the lipid products of this enzyme system, such as PtdIns(3,4,5)P3 also do not interact directly with KATP channels [22]. Recent studies demonstrate that both leptin and PtdIns(3,4,5)P3 increase KATP channel activity indirectly, through changes in cytoskeletal dynamics [31]. It is well established that many ion channels and transporters are anchored in the membrane by either direct or indirect association with the cytoskeleton. In addition, there is growing evidence that altering the integrity of cytoskeletal elements, in particular actin filaments, can modulate the activity of a variety of ion channels [40] and receptors [41]. For example, disruption of actin filaments with cytochalasin is shown to increase KATP channel activity in cardiac myocytes [42] and CRI-G1 cells [31]. Indeed, a number of lipid kinases, including PI 3-kinase, are also localised to the cytoskeleton and their activities are modulated by a variety of cytoskeletal proteins, especially those associated with actin [40]. Actin filament structure is controlled by reversible polymerisation of G-actin, which forms F-actin, and this process is under the dynamic control of various actin-binding proteins [43]. The heptapeptide mushroom toxin phalloidin [32] binds to filamentous F-actin with high affinity and stabilises the actin in this form. The addition of phalloidin to the intracellular aspect of CRI-G1 cells prevented BVT.12777, but not diazoxide, from activation of KATP channel currents in whole cell and inside out recording configurations indicating that this molecule likely causes the opening of KATP channels by a membrane delimited alteration of cytoskeletal dynamics. This mechanism of action is identical to that proposed for leptin and PtdIns(3,4,5)P3 activation of KATP in this cell line [31]. Fluorescence staining of CRI-G1 cells with rhodamine-conjugated phalloidin revealed disassembly of actin filaments by both BVT.12777 and leptin, but not diazoxide. These data provide direct support for an important role for cytoskeletal dynamics in the control of KATP channel activity by both leptin and BVT.12777. The lack of effect of diazoxide on the actin filament structure is also supportive of this opener acting directly on the KATP channel subunits. Conclusions BVT.12777 activation of KATP channels in CRI-G1 cells was evident regardless of whether it was applied to the external or internal surface of the cell. BVT.12777 signalling to KATP channels is not mediated by PI 3-kinase or MAPK, but does appear to depend on actin filament re-modelling. As leptin hyperpolarizes a sub-population of hypothalamic neurones by opening KATP channels [16], it is feasible that at least part of the anti-obesity action of BVT.12777 may be through the activation of this potassium channel. Furthermore, as BVT.12777 acts downstream of PI3K, such an agent may act to overcome the putative central leptin resistance associated with the obese state [37]. Thus, although BVT.12777 and its close structural analogues are unlikely per se to be useful anti-obesity agents as they display hepatotoxicity [44], understanding the general principles underlying their mechanism of action may reveal clues for future anti-obesity drug development. Methods Cell culture and transfection Cells from the insulin secreting cell line, CRI-G1, and the human embryonic kidney cell line, HEK 293, were grown as described previously [25,35]. The preparation of mouse Kir6.2 (provided by Professor F. Ashcroft, University of Oxford), rat SUR 1 (provided by Dr G. Bell, University of Chicago) and CD4 cDNAs and transfection procedures were as described by [25]. Transfected cells were selected by visible binding of anti-CD4 coated beads (Dynal, Oslo) following incubation with the beads for 20 min. Oocyte collection and preparation Ovarian lobes were removed from mature female Xenopus laevis frogs (Blades Biological, UK) following killing of the animal by destruction of the brain. The use of animals was in accordance with the Home Office Animals (Scientific Procedures) Act (1986) and approved by the local ethics committee. Separation and selection of oocytes and the preparation and injection of cRNAs were performed as described by [25]. Western blotting CRI-G1 cells, in normal saline (containing in mM; NaCl 135, KCl 5, MgCl2 1, CaCl2, 1, HEPES 10 with glucose 10 (pH 7.4) were treated with BVT.12777 (100 μM) for 0, 1, 5, 15 or 30 minutes and whole-cell extracts were prepared as described [23]. Proteins (10 μg) were suspended in loading buffer (Invitrogen) and after denaturation, loaded on to NuPage 4–12% Bis-Tris mini-gels (Invitrogen) and run at 200 V for 1 hr. Subsequently, proteins were transferred to Hybond-C Extra nitrocellulose membranes (Amersham) at 25 V for 80 minutes at room temperature. Membranes were incubated in blocking buffer (5% non-fat milk in TBST (20 mM Tris HCl, 150 mM NaCl, 0.5% Tween, pH 7.4)) for 1 hr at room temperature after which antibodies to phospho-MAPK, phospho-STAT3, phospho PKB, phospho-GSK3 and PKB (all at 1:1000) were applied at 4°C with gentle shaking, overnight. The membranes were washed with TBST (4 × 30 minutes) and incubated for 1 hr at room temperature with HRP conjugated ImmunoPure goat anti-rabbit IgG (1:5000). After washing with TBST (5 × 15 minutes), immunoreactive bands were visualised by the enhanced chemiluminescence (ECL) detection reagent (Amersham). Cytoskeletal fluorescence imaging and analysis CRI-G1 cells were gently washed in normal saline (containing in mM): NaCl 135, KCl 5, MgCl2 1, CaCl2 1, HEPES 10, pH 7.4, and incubated for 40 min with either 100 μM BVT.12777, 10 nM leptin, 200 μM diazoxide or 3 mM sodium azide for 30 min with the cytoskeletal disrupter, cytochalasin B (10 μM). Cells were then fixed, permeabilised, stained with rhodamine-conjugated phalloidin (2.66 U ml-1) and visualised using a BioRad Microradiance, confocal imaging system as described by [31]. The intensity of rhodamine-conjugated phalloidin staining in the plasma membrane was determined using BioRad Lasersharp processing software (Bio-Rad, CA, USA). Analysis lines were drawn along randomly selected regions of the plasma membrane and the fluorescence intensity determined. A histogram giving the mean fluorescence intensity was constructed for a minimum of 5 cells on each stimulated or control dish on at least 3 separate occasions. Within a given experimental series all conditions for capturing images were constant. In order to allow for quantification of experimental data obtained on separate days, the results were normalised relative to the mean plasma membrane fluorescence measured in the control cells for each day and presented as mean ± S.E.M. Statistical analyses were performed using Student's unpaired t test. p < 0.05 was considered significant. Electrophysiological recording and analysis Whole cell currents from Xenopus oocytes were measured using a two-electrode voltage clamp technique as described by [25]. Recordings were made in a high-potassium bath solution, KD96 containing (mM): KCl 96, NaCl 2, CaCl2 1.8, HEPES 5 (pH 7.4 with KOH). Working concentrations of drugs were prepared in KD96 and superfused into the bath. Whole-cell current-clamp recordings with excursions to voltage clamp mode were used to monitor membrane potential and macroscopic currents from CRI-G1 cells. Cell-attached and excised inside-out recordings were made from CRI-G1 cells and HEK cells expressing Kir6.2 and SUR1 to examine single channel responses as described previously [25,35]. Single channel data were analysed for current amplitude and channel activity (Nf.Po; where Nf is the number of functional channels in the patch and Po is the open probability) as described previously [45]. All data were normalised to control and are expressed as mean ± S.E.M. Statistical analyses were performed using Student's unpaired t test. P < 0.05 was considered significant. Recording electrodes were pulled from borosilicate glass and had resistances of 2–5 MΩ for whole cell recordings and 7–10 MΩ for cell-attached and inside-out experiments when filled with electrolyte solution. The pipette solution for whole-cell recordings comprised (in mM): KCl 140, MgCl2 0.6, CaCl2 2.73, Mg-ATP 5.0, EGTA 10, HEPES 10, pH 7.2 (free [Ca2+] of 100 nM), whereas for single channel recordings the pipette solution contained (in mM): KCl 140, CaCl2 1, MgCl2 1, HEPES 10, pH 7.2. The bath solution for whole-cell and cell-attached recordings was normal saline whereas for inside-out patches the bath solution contained (in mM): KCl 140, MgCl2 1, CaCl2 2, EGTA 10, HEPES 10, pH 7.2 (free [Ca2+] of 30 nM). All solution changes were achieved by superfusing the bath with a gravity feed system at a rate of 10 ml min-1, which allowed complete exchange within 2 min. All experiments were performed at room temperature (22–25°C). Antibodies & drugs Anti-PKB, which recognises all three isoforms of PKB, and the phospho-specific PKB (Thr308), GSK3α/β (Ser21/9), STAT3 (Tyr705) and p44/42 MAPK (Thr202/Tyr204) antibodies were obtained from Cell Signalling Technology Inc. Recombinant human leptin, wortmannin and LY 294002 were obtained from Novachem-Calbiochem and BVT.12777 ([2-(hydrazinoiminomethyl) hydrazino] acetic acid) was a gift from Biovitrum (Stockholm, Sweden). Tolbutamide, Mg-ATP, diazoxide, sodium azide, phalloidin and cytochalasin B were obtained from Sigma. Rhodamine-conjugated phalloidin was obtained from Molecular Probes and UO126 from Promega. BVT.12777 was prepared as a 100 mM stock solution in normal saline and stored at -70°C prior to use. Leptin was prepared as a 10 μM stock solution in normal saline containing 0.2 % bovine serum albumin as carrier. Rhodamine-conjugated phalloidin (200 U ml-1) and LY 294002 (10 mM) were stored as stock solutions in 1% methanol at -20°C. Cytochalasin B was stored as a 10 mM stock solution, and diazoxide and tolbutamide as 100 mM solutions, all in DMSO at 2–4°C. Mg-ATP was stored at -20°C as a 100 mM solution in 10 mM HEPES (pH 7.2). Wortmannin and UO126 were stored as 10 mM stock solutions in Me2SO at -20°C. List of abbreviations used CRI-G1, Cambridge Rat Insulinoma-G1; GSK3, glycogen synthase kinase-3; HEK293, human embryonic kidney 293; JAK, janus kinase; KATP, ATP-sensitive potassium; Kir6.2, potassium channel inward rectifier-6.2; MAPK, p42, p44 mitogen-activated protein kinase; MAPKK, MAPK kinase; ObRb, Obese (leptin) receptor-b; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5 tris-phosphate; STAT3, signal transducer and activator of transcription-3; SUR, sulphonylurea receptor Authors' contributions JK carried out the majority of the electrophysiology and cytoskeletal fluorescence studies. HL carried out the western blot experiments. TT and JH participated in the electrophysiological experiments. CS participated in the design and implementation of the western blot experiments. MA conceived of the study, participated in its design and co-ordination and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported in part by grants from The Wellcome Trust (042726 & 065287), Biovitrum AB and Tenovus, Scotland. CS is a Diabetes UK Senior Fellow. ==== Refs Seino S ATP-sensitive potassium channels: a model of heteromultimeric potassium channel/receptor assemblies Annu Rev Physiol 1999 61 337 362 10099692 10.1146/annurev.physiol.61.1.337 Babenko AP Aguilar-Bryan L Bryan J A view of SUR/Kir6.x, KATP channels Annu Rev Physiol 1998 60 667 687 9558481 10.1146/annurev.physiol.60.1.667 Ashcroft FM Gribble FM ATP-sensitive K+ channels and insulin secretion: their role in health and disease Diabetologia 1999 42 903 919 10491749 10.1007/s001250051247 Lawson K Potassium channel openers as potential therapeutic weapons in ion channel disease Kidney Int 2000 5 838 845 10720937 10.1046/j.1523-1755.2000.00923.x Gribble FM Reimann F Pharmacological modulation of KATP channels Biochem Soc Trans 2002 30 333 339 12023875 10.1042/0300-5127:0300333 Rorsman P The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint Diabetologia 1997 40 487 495 9165215 10.1007/s001250050706 Proks P Reimann F Green N Gribble FM Ashcroft FM Sulfonylurea stimulation of insulin secretion Diabetes 2002 51 S368 S376 12475777 Dunne MJ Ions, genes and insulin release: from basic science to clinical disease Diabetic Med 2000 17 91 104 10746478 10.1046/j.1464-5491.2000.00247.x Babenko AP Gonzalez G Bryan J Pharmaco-topology of sulfonylurea receptors: separate domains of the regulatory subunits of KATP channel isoforms are required for selective interaction with K+ channel openers J Biol Chem 2000 275 717 720 10625598 10.1074/jbc.275.2.717 Dabrowski M Ashcroft FM Ashfield R Lebrun P Pirotte B Egebjerg J Bondo Hansen J Wahl P The novel diazoxide analog 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide is a selective Kir6.2/SUR1 channel opener Diabetes 2002 51 1896 1906 12031979 Mannhold R KATP channel openers: structure activity relationships and therapeutic potential Med Res Rev 2004 24 213 266 14705169 10.1002/med.10060 Kieffer TJ Heller RS Leech CA Holz GG Habener JF Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells Diabetes 1997 46 1087 1093 9166685 Harvey J Mckenna F Herson PS Spanswick D Ashford MLJ Leptin activates ATP-sensitive potassium channels in the rat insulin-secreting cell line, CRI-G1 J Physiol 1997 504 527 535 9401961 Kieffer TJ Habener JF The adipoinsular axis: the effects of leptin on pancreatic β-cells Am J Physiol Endocrinol Metab 2000 278 E1 E14 10644531 Ahima RS Flier JS Leptin Annu Rev Physiol 2000 62 413 437 10845097 10.1146/annurev.physiol.62.1.413 Spanswick D Smith MA Groppi V Logan SD Ashford MLJ Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels Nature 1997 390 521 525 9394003 10.1038/37379 Lee K Dixon AK Richardson PJ Pinnock RD Glucose-receptive neurons in the rat ventromedial hypothalamus express KATP channels composed of Kir6.1 and SUR1 subunits J Physiol 1999 515 439 452 10050011 Miki T Liss B Minami K Shiuchi T Saraya A Kashima Y Horiuchi M Minokoshi Y Roeper J Seino S ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis Nature Neurosci 2001 4 507 512 11319559 Meglasson MD Wilson JM Yu JH Robinson DD Wyse BM Desousa CJ Antihyperglycemic action of guanidine-alkanoic acids: 3-guanidinopropionic acid ameliorates hyperglycemia in diabetic KKAy and C75BL6J ob/ob mice and increases glucose disappearance in rhesus monkeys J Pharmacol Exp Ther 1993 266 1454 1462 8371149 Vaillancourt VA Larsen SD Tanis SP Burr JE Connell MA Cudahy MM Evans BR Fisher PV May PD Meglasson MD Robinson DD Stevens FC Tucker JA Vidmar TJ Yu JH Synthesis and biological activity of aminoguanidine and diaminoguanidine analogues of the antidiabetic/antiobesity agent 3-guanidinopropionic acid J Med Chem 2001 44 1231 1248 11312923 10.1021/jm000094n Carrington CA Rubery ED Pearson EC Hales CN Five new insulin-producing cell lines with differing secretory properties J Endocrinol 1986 109 193 200 3011941 Harvey J Mckay NG Van Der Kaay J Downes CP Ashford MLJ Essential role of phosphoinositide 3-kinase in leptin-induced KATP channel activation in the rat CRI-G1 insulinoma cell line J Biol Chem 2000 275 4660 4669 10671495 10.1074/jbc.275.7.4660 Harvey J Ashford MLJ Insulin occludes leptin activation of ATP-sensitive K+ channels in rat CRI-G1 insulin secreting cells J Physiol 1998 511 695 706 9714853 Gribble FM Ashfield R Ammala C Ashcroft FM Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes J Physiol 1997 498 87 98 9023770 McKay NG Kinsella JM Campbell CM Ashford MLJ Sensitivity of Kir6.2-SUR1 currents, in the absence and presence of sodium azide, to the KATP channel inhibitors, ciclazindol and englitazone Br J Pharmacol 2000 130 857 866 10864893 Alessi DR Cuendo A Cohen P Dudley DT Saltiel AR PD 98059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo J Biol Chem 1995 270 27489 27494 7499206 10.1074/jbc.270.46.27489 Lazar DF Wiese RJ Brady MJ Mastick CC Waters SB Yamauchi K Pessin JE Cuatrecasas P Saltiel AR Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin J Biol Chem 1995 270 20801 20807 7657664 10.1074/jbc.270.35.20801 Takahashi Y Okimura Y Mizuno I Takahashi T Kaji H Uchiyama T Abe H Chihara K Leptin induces mitogen-activated protein kinase dependent proliferation of C1H10T1/2 cells J Biol Chem 1997 272 12897 12900 9148892 10.1074/jbc.272.20.12897 Tanabe K Okuya S Tanizawa T Matsutani A Oka Y Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein kinase Biochem Bioph Res Com 1997 241 765 768 10.1006/bbrc.1997.7894 Davies SP Reddy H Caivano M Cohen P Specificity and mechanism of action of come commonly used protein kinase inhibitors Biochem J 2000 351 95 105 10998351 10.1042/0264-6021:3510095 Harvey J Hardy SC Irving AJ Ashford MLJ Leptin activation of ATP-sensitive K+ (KATP) channels in rat CRI-G1 insulinoma cells involves disruption of the actin cytoskeleton J Physiol 2000 527 95 107 10944173 10.1111/j.1469-7793.2000.00095.x Wieland I Faulstich H Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms Crit Rev Biochem 1978 5 185 260 363352 Cooper JA Effects of cytochalasin and phalloidin on actin J Cell Biol 1987 105 1473 1478 3312229 10.1083/jcb.105.4.1473 Kozlowski RZ Hales CN Ashford MLJ Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line Br J Pharmacol 1989 97 1039 1050 2676059 Harvey J Hardy SC Ashford MLJ Dual actions of the metabolic inhibitor, sodium azide on KATP channel currents in the rat CRI-G1 insulinoma cell line Br J Pharmacol 1999 126 51 60 10051120 Sturgess NC Kozlowski RZ Carrington CA Hales CN Ashford MLJ Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line Br J Pharmacol 1988 95 83 94 3146398 Sweeney G Leptin signalling Cell Signal 2002 14 655 663 12020765 10.1016/S0898-6568(02)00006-2 Niswender KD Schwartz MW Insulin and leptin revisited: adiposity signals with overlapping physiological and intracellular capabilities Front Neuroendocrinol 2003 24 1 10 12609497 10.1016/S0091-3022(02)00105-X Gribble FM Tucker SJ Ashcroft FM The essential role of the walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide EMBO J 1997 16 1145 1152 9135131 10.1093/emboj/16.6.1145 Janmey PA The cytoskeleton and cell signalling: component localisation and mechanical coupling Ann Rev Physiol 1994 56 169 191 8010739 Wang H Bedford FK Brandon NJ Moss SJ Olsen RW GABAA-receptor-associated proteins links GABAA receptors and the cytoskeleton Nature 1999 397 69 72 9892355 10.1038/16264 Terzic A Kurachi Y Actin microfilament disrupters enhance K (ATP) channel opening in patches from guinea-pig cardiomyocytes J Physiol 1996 492 395 404 9019537 Pollard TD Borisy GG Cellular motility driven by assembly and disassembly of actin filaments Cell 2003 112 453 465 12600310 10.1016/S0092-8674(03)00120-X Schuppe-Koistinen I Frisk AL Janzon L Molecular profiling of hepatotoxicity induced by a aminoguanidine carboxylate in the rat: gene expression profiling Toxicology 2002 179 197 219 12270593 10.1016/S0300-483X(02)00355-4 Lee K Rowe ICM Ashford MLJ Characterisation of an ATP-modulated large conductance Ca2+-activated K+ channel present in rat cortical neurones J Physiol 1995 488 319 337 8568673

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==== Front BMC Plant BiolBMC Plant Biology1471-2229BioMed Central London 1471-2229-4-151533101910.1186/1471-2229-4-15Research ArticleCloning and expression analysis of cDNAs corresponding to genes activated in cucumber showing systemic acquired resistance after BTH treatment Bovie Catherine [email protected] Marc [email protected] Philippe [email protected] Jacques [email protected] Laboratoire de Biologie Moléculaire et de Biotechnologie Végétales, Département des Sciences de la Vie, B22, Université de Liège, B-4000 Liège/Sart Tilman, Belgium2 Centre Wallon de Biologie Industrielle, Unité de Bioindustries, Faculté Universitaire des Sciences Agronomiques, B-5030 Gembloux, Belgium2004 26 8 2004 4 15 15 18 5 2004 26 8 2004 Copyright © 2004 Bovie et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Infection of plants by necrotizing pathogens can lead to the rapid and localized induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. Subsequently, an increase of plant resistance against a broad spectrum of pathogens is observed systemically. This plant immunity is known as Systemic Acquired Resistance. To identify components of the transduction pathway, we cloned and analysed the expression pattern of several mRNAs accumulating in cucumber plants after induction of Systemic Acquired Resistance. Results We tested on cucumber different compounds known to induce systemic acquired resistance. Among these, BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) proved to be very effective. mRNA RT-PCR differential display was used to identify mRNA sequences induced 24 hours after the application of 10 μM BTH to cucumber plants. A cDNA library constructed from cucumber plants sprayed with 10 μM BTH was screened to get corresponding full length cDNAs. Among the identified cDNAs were those coding for a putative ras-related GTP-binding protein, a putative beta-1,4-N-Acetylglucosaminyltranferase III and a putative pathogenesis related protein. The time course of accumulation of the three corresponding mRNAs was analysed by northern blotting in plants treated by BTH or in plants infected by Colletotrichum lagenarium. Conclusions The mRNA RT-PCR differential display technique allowed the identification of three genes possibly involved in Systemic Acquired Resistance in cucumber. Pathogenesis-related proteins are known to be involved in plant defence against pathogens. GTP-binding protein and N-acetylglucosaminyltranferase III have been reported to be components of signal transduction pathways in mammals and plants. ==== Body Background Infection of plants by necrotizing pathogens can lead to the induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. The infected leaves develop a hypersensitive response (HR), i.e., rapid, localized cell death occurring at the sites of pathogen entry [1]. Concomitant with the HR is the accumulation of salicylic acid and several classes of pathogenesis-related (PR) proteins, many of which exhibiting antimicrobial activity [2]. Subsequently, an enhancement of the plant defensive capacity against a broad spectrum of pathogens is observed. This resistance is expressed locally as well as in distal, uninfected tissues and can last for several weeks to months. It is known as Systemic Acquired Resistance or SAR [3]. Salicylic acid was identified as an important signal in the SAR transduction pathway. It was shown to accumulate during the onset of SAR in cucumber [4], tobacco [5], and Arabidopsis thaliana [6]. Exogenously supplied salicylic acid induces the same set of genes and resistance against the same spectrum of pathogens, as with pathogen-induced SAR [3]. Plants in which salicylic acid accumulation is prevented by over expression of a bacterial salicylate hydroxylase gene failed to develop SAR and/or exhibited increased susceptibility to pathogen infection [7,8]. Analysis of A. thaliana mutants has revealed that the NPR1 protein (also known as NIM1) is required for SAR induction and acts downstream of salicylic acid in the SAR pathway. Plants carrying mutations in this gene fail to express several PR genes and display enhanced susceptibility to infection [9,10]. The NPR1/NIM1 protein interacts with members of the TGA/OBF family of basic leucine zipper transcription factors. Some of these factors have been shown to bind to salicylic acid-responsive promoter elements of the PR-1 gene [11-13]. The salicylic acid induced interaction of NPR1 with TGA factors is localized in the nucleus [14], while cytoplasmic NPR1 appears to modulate crosstalk between salicylic acid- and jasmonate-dependent defense pathways [15]. Other components of the salicylic acid signaling pathway have been identified using genetic approaches in Arabidopsis thaliana [16]. They include PAD4 and EDS3 which activate EDS4, SID2 and EDS5 leading to salicylic acid accumulation [17]. Besides pathogens, SAR is also induced by exogenously applied chemicals. In addition to salicylic acid, well-known chemical inducers of PR genes include 2,6-dichloroisonicotinic acid (INA), benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) and L-α-amino butyric acid (ABA) [18-23]. SAR induction appears to be governed by a complex signal transduction process [24] that involves a signal originating at the site of infection or treatment, and moving throughout the plant. Chemical inducers provide tools for dissection of the signal transduction pathway that regulates the defense responses (they enter the pathway at different points). On another hand, the cucumber plant provides a good model system for studying induced disease resistance [25]. Local and systemic increases in chitinase and/or peroxidase activity have been observed in response to inoculation with necrotizing pathogens or to treatment with SA, INA and BTH [26-28]. The aim of the present work was to identify and clone mRNAs accumulating after induction of SAR in cucumber plants. These sequences could correspond to proteins potentially involved in the signal transduction pathway leading to SAR or to proteins responsible for the state of resistance. In a first set of experiments, potential SAR inducing chemicals were tested in order to set up a reproducible system of activation of this resistance in cucumber. The mRNA RT-PCR differential display method [29] was then used to identify genes activated during SAR induction. Several differentially expressed cDNA were identified and some of them were cloned and sequenced. Their expression was analyzed by Northern blotting. Results Chemically induced resistance in cucumber The SAR inducing activity of different chemicals has already been documented in one or several plant species [18-23]. To evaluate their activity on cucumber, cotyledons of two-week-old plants were infiltrated with 0.5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine or 20 mM BaCl2. Control plants were infiltrated with water. In a parallel experiment, four-week-old cucumber plants (with two true leaves) were sprayed with 5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine, 20 mM BaCl2 or 1 mM BTH. Control plants were sprayed with water or left untreated. Total RNAs were extracted from treated leaves 6 h, 24 h or 48 h after treatment. Induction of PR-8 gene was assayed by Northern blotting followed by hybridization with a cucumber PR-8 cDNA probe. PR-8 cucumber gene encodes an acidic chitinase which accumulates after tobacco necrosis virus (TNV) infection or salicylic acid induction and is considered as a SAR molecular marker [30]. In infiltration experiments (results not shown), control plants showed positive hybridization signals. To check whether this activation of the PR-8 gene could be due to wounding during infiltration, the effect of wounding alone was evaluated by making a cut from the midvein to the outer edge of the cotyledons. A strong signal was obtained after wounding which could be explained by induction of the acidic chitinase itself or of an isoform. Therefore infiltration did not prove to be an appropriate method for applying SAR inducers in cucumber, because it probably induced a wound response. When applied by spraying, BTH, L-α-amino butyric acid and 2-thiouracil strongly induced PR-8 mRNA accumulation, while SA treatment led to a lower induction level (Fig. 1). Thiamine and BaCl2 induced slightly PR-8 expression (results not shown). RNA from control plants did not show any hybridization signal indicating that spraying itself did not induce PR-8 expression. BTH, which is an analogue of salicylic acid, showed the strongest PR-8 induction. But cucumber plants treated with 1 mM BTH showed a reduced growth (smaller leaves area and shorter internodes). As BTH was shown to be a very efficient activator of disease resistance in different plant species [20-23], we tried to induce SAR in cucumber plants with lower BTH concentrations (10 μM, 30 μM and 100 μM). PR-8 transcript accumulation was quite similar for the three concentrations (see Additional file 1) and no adverse effect on plant growth was observed. In cucumber, SAR has been shown to be effective against anthracnose caused by Colletotrichum lagenarium and salicylic acid treatment was shown to induce resistance against this pathogen [31]. To confirm that BTH acts as a chemical inducer of SAR in cucumber, four-week-old plants were sprayed with water, 10 μM BTH, 50 μM BTH, or left untreated, 5 days prior to inoculation of the second true leaf with C. lagenarium. Two weeks after inoculation, the plants were scored for spreading lesions, i.e. lesions showing a larger size than the initial inoculated area. As shown in figure 2 and table 1, BTH treatment resulted in decreased symptoms. Control leaves inoculated with C. lagenarium showed large spreading lesions and thus extensive fungal growth. Treatment of plants with BTH prior to C. lagenarium inoculation resulted in a markedly decreased lesion formation (Fig. 2). In untreated plants and water treated plants, 67 and 64 % of inoculation sites showed spreading lesions respectively (Table 1). In BTH treated plants, only 23 % (10 μM BTH) or 9 % (50 μM BTH) of inoculated sites developed spreading lesions (Table 1). To check whether BTH is able to act systemically or is transported through cucumber plants, the third leaf of four-week-old plants was wrapped with a plastic film and the plants were sprayed with H2O, 10 μM BTH, 50 μM BTH or left untreated. This leaf was inoculated with C. lagenarium 7 days later. The percentage of spreading lesions on third leaves from pathogen-challenged untreated or H2O treated cucumber plants (infected controls) were respectively 86% and 83.5%. In BTH treated plants, 38 % (10 μM BTH) or 12 % (50 μM BTH) of inoculated sites developed spreading lesions (Table 2). These results suggest that BTH can activate resistance in untreated leaves. Differential expression of genes in BTH induced cucumber plants Plants were sprayed with 10 μM BTH or water. Sampling was performed 24 h after treatment in order to point out early-induced differences in gene expression. RT-PCR products were separated on polyacrylamide gels. Bands that appeared on the display of RNA only from BTH treated plants, or only from water treated plants were likely to correspond to differentially expressed mRNAs. Twelve differentially expressed bands were identified (Table 3). Their sizes ranged between 250 and 450 bp. They corresponded to genes expressed in the BTH treated plants and not in the control ones or presenting a higher expression in the BTH treated plants compared to the control ones (Fig. 3). These bands were eluted from the gel, PCR amplified and cloned in the pGEM-T vector. In order to confirm the differential expression of these genes, reverse slot blot hybridizations were performed: DNA from four to ten clones obtained from each differentially displayed band was fixed on a membrane and hybridized with the corresponding reverse transcribed products from either BTH or H2O treated plants (Fig. 4). Six from the twelve fragments were confirmed to be differentially expressed: dd3, dd4, dd5, dd6, dd7 and dd11. Five bands (dd1, dd8, dd9, dd10 and dd12) were either expressed evenly between BTH and H2O treated plants (differential display false positives or signal masked by the expression of homologous genes). In the remaining case (dd2), no signal could be observed on the slot blots. This absence of hybridization signal could be due to a lack of sensitivity of the slot blot hybridization procedure. The dd3, dd4, dd5, dd6, dd7 and dd11 cDNA fragments were sequenced. Blast analysis did not lead to identification of any homology with known sequences. This is not surprising as mRNA differential display generates fragments corresponding to 3'untranslated regions for which homologies are difficult to find. Two of the six fragments were proved to be part of the same gene (dd5 and dd7). A cDNA library was constructed from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment). The library was screened with the five differentially expressed fragments. Hits were obtained for four of these probes (corresponding to dd3, dd4, dd6 and dd11). Full length cDNAs corresponding to dd4, dd6 and dd11 were obtained. Homology between cloned cDNA and sequences in databases is presented in table 4. The cDNA dd3 matched to a putative RAS-related GTP-binding protein from A. thaliana. It will be referred to as CRG (cucumber RAS-related GTP-binding protein). The cDNA dd4 matched to a translation releasing factor 2. It will be referred to as CTR (cucumber translation releasing factor 2). The cDNA dd6 matched to a putative β-1,4-N-Acetylglucosaminyltranferase III from A. thaliana. This protein was named CGT (cucumber acetylglucosaminyltransferase III). The cDNA dd11 matched to a putative pathogenesis related protein from A. thaliana and, with slightly less homology, to a pathogenesis related protein from barley induced by fungal infection [32]. It will be referred to as CPR (cucumber pathogenesis-related protein). PR-8, CPR, CRG and CGT expression analysis To investigate the expression of PR-8, CPR, CRG and CGT genes in cucumber, the two first leaves of four-week old plants were sprayed with 10 μM BTH. In another batch of plants, the first leaf was inoculated with C. lagenarium. Total RNA was extracted from treated leaves or upper untreated leaves at various times after treatment. Accumulation of the PR-8, CPR, CRG and CGT mRNAs was assayed by Northern blotting followed by hybridization with the corresponding cDNA probes (fig. 5). Expression of PR-8 gene was induced at a high level by both 10 μM BTH spraying and C. lagenarium infection in the leaves submitted to the treatment. PR-8 mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 72 h. PR-8 expression was observed as from 72 h after inoculation with C. lagenarium. No significant PR-8 mRNA accumulation could be detected in the untreated leaves from BTH sprayed plants, while in non infected leaves PR-8 expression was observed 72 h after infection by C. lagenarium; the level of expression was still increasing 2 weeks after C. lagenarium inoculation. CPR gene expression was strongly induced in both 10 μM BTH treated and C. lagenarium inoculated leaves. CPR mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 24 h. CPR expression was observed 72 h after infection by C. lagenarium. No expression of CPR gene could be observed in untreated leaves from BTH sprayed plants, while CPR mRNA accumulation could be detected in uninfected leaves from C. lagenarium infected plants 72 h after inoculation. CGT gene showed a significant basal level of expression as it could be observed in control plants (fig. 5). Nevertheless the expression of CGT was increased both by spraying with 10 μM BTH (with a maximum at 24 h) and by inoculation with C. lagenarium (with a maximum at 72 h) in the leaves submitted to the treatment. A systemic induction could also be observed in untreated leaves from C. lagenarium inoculated plants (as from 72 h after infection). The expression level of the CRG gene was too low to allow us to show an induction of the expression (data not shown). Expression of the same genes was analyzed in wounded cucumber plants. A cut was made from the midvein to the outer edge of the first leaf. Total RNA was extracted from the cut leaf or the upper leaf, 24 and 48 h after wounding. An induction of the expression of PR-8 and CPR genes was only observed in the cut leaf 24 h after wounding. No expression was observed later nor in the uncut upper leaf. The CGT and CRG genes were not induced by wounding (results not shown). Expression of these genes was also analyzed in cucumber plants showing rhizobacteria-induced systemic resistance. Plants were grown in compost drenched with a Pseudomonas putida BTP1 suspension, a plant growth-promoting rhizobacteria known to promote induced systemic resistance in cucumber [33]. Total RNA was extracted from cotyledons collected three weeks after sowing. PR-8, CPR, CGT and CRG did not show any induction of expression in these conditions (results not shown). Discussion We tested here two different techniques and several compounds to induce SAR in cucumber. The expression of PR-8 gene, which encodes an acidic chitinase from cucumber, was used as an indicator of the activation of the defense response. The accumulation of this acidic chitinase after tobacco necrosis virus infection or salicylic acid induction correlates with induced resistance [30]. Among the different compounds tested, BTH applied by spraying proved to be the stronger inducer of PR-8 mRNA accumulation. BTH sprayed at a concentration of 10 μM was still able to induce PR-8 gene expression. The protective effects of BTH on cucumber were confirmed in challenge experiments with C. lagenarium. Spraying cucumber plants with BTH (10 μM or 50 μM) allowed effective local and systemic protection against C. lagenarium. BTH was recently identified as a safe, reliable and non phytotoxic plant protection agent by scientists at Novartis. Exogenous application of BTH to tobacco, A. thaliana, wheat and cucumber has been shown to activate a number of SAR associated genes leading to enhanced plant protection against various pathogens [20-22,28]. Identifying SAR induced genes could provide clues to elucidate the signal transduction pathway leading to plant defense responses. We used RT-PCR differential display in BTH-treated cucumber plants to detect SAR associated mRNAs. We have identified four BTH-inducible genes: a putative ras-related GTP binding protein (CRG), a putative translation releasing factor 2 (CTR), a putative β-1,4-N-acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Expression of PR-8, CPR and CGT genes was shown to be induced by 10 μM BTH treatment as well as C. lagenarium infection. The response to BTH was observed earlier (5 h after treatment) than the one developed after pathogen inoculation (72 h after infection). However, systemic expression of these genes was only observed in C. lagenarium infected plants. Small GTP-binding proteins form a large family of nucleotide triphosphatases whose activity is related to the binding, hydrolysis and release of guanosine triphosphate [34]. MAP kinase cascade represents an important downstream effector pathway for RAS in most cells [35] and recent studies have shown that MAP kinases are activated in plants in response to pathogen attack and wounding [36,37]. Small GTP-binding proteins have been shown to be involved in important cell mechanisms like cell division, transduction of external signals across the plasma membrane, endocytosis and exocytosis, cell death in plants and establishment of plant defense reaction [34,38-42]. The β-1,4-N-Acetylglucosaminyltranferase III catalyses the addition of N-acetylglucosamine to the β-mannoside of the tri-mannose core in N-glycans, resulting in the suppression of further processing and elongation. In mammals, N-acetylglucosaminyltransferase III expression is associated with differentiation, cell adhesion and tumor progression [43,44]. It was shown recently to interfere with epidermal growth factor signaling and H2O2-induced activation of protein kinase C [45,46]. N-linked glycans were shown to be widely distributed in plants and highly expressed at the cell surface, which might suggest a putative function in cell/cell communication [47]. To our knowledge β-1,4-N-Acetylglucosaminyltranferase has never been described in the context of a plant-pathogen interaction. Several cDNAs or gene encoding enzymes involved in the biosynthesis of complex N-linked glycans have been cloned from plants. They include N-acetylglucosaminyltranferase I cDNAs from A. thaliana, potato and tobacco [48], N-acetylglucosaminyltransferase II cDNA from A. thaliana [49] and defense-related glucosyl transferase gene from tomato [50]. GTP-binding proteins and N-acetylglucosaminyltranferases are involved in important mechanisms, notably signaling pathways and therefore seem of high interest. The putative PR protein, which has been first identified in barley after fungal infection [32] has no known function. Only PR-8 and CPR genes were induced by wounding but not systemically. PR-8 and CPR genes were not induced by P. putida BTP1 treatment, which triggers induced systemic resistance (ISR) in cucumber. ISR is phenotypically similar to SAR but the mechanisms of this resistance were shown to be different: although this was not tested with P. putida BTP1, in other experimental systems ISR was shown to be dependent on NPR/NIM1 function, but does not involve salicylic acid, nor PR protein accumulation [51]. Our results correlate with those of Pieterse et al. who showed that induced systemic resistance in A. thaliana was independent of PR genes expression [51]. In the same way, ISR also seems to be independent on CGT and CRG expression, as these genes were not induced by P. putida BTP1. Conclusions Among different SAR inducers tested on cucumber, BTH proved to be very efficient. Applied by spraying, BTH induced PR-8 mRNA accumulation and allowed effective local and systemic protection against C. lagenarium. Using RT-PCR differential display, we identified four BTH-inducible genes, including a putative ras-related GTP binding protein (CRG), a translation releasing factor 2 (CTR), a putative β-1,4-N-acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Genes expression studies confirmed the local induction of CGT and CPR after BTH treatment or C. lagenarium infection and their systemic induction in response to C. lagenarium infection. Moreover CPR was locally induced by wounding. The significant level of expression of CGT and CPR genes during defense responses suggests a potential role for the gene products in the SAR pathway or in the state of resistance in cucumber. Methods Organisms and growth conditions Cucumber plants (Cucumis sativus cv. Marketer) were grown in compost at 22°C with a 16 h light photoperiod. Cultures of Colletotrichum lagenarium were maintained on malt agar at 25 °C. Spores suspensions in water supplemented with 0.01 % tween 80 were prepared from four- to six-week-old cultures grown in petri plates. Suspensions were filtered through cheesecloth and the spore concentration determined with a hemacytometer. Chemical treatments All chemicals were obtained from Sigma except for BTH (provided by Novartis). Chemicals were dissolved in water and the pH adjusted to 6.6 with NaOH except for thiamine, BaCl2 and BTH. Infiltration experiments: cotyledons of two-week-old plants were infiltrated through a vein until the whole leaf was impregnated with the solution. Spraying experiments: the two first true leaves of four-week-old cucumber plants were sprayed with +/- 3 ml solution/plant. The upper leaves were protected with a plastic sheet when systemic resistance was to be tested. ISR induction Prior to planting, disinfected cucumber seeds were soaked for 10 min in a Pseudomonas putida BTP1 suspension (4.108 CFU/ml in 0.85% NaCl). Control seeds were soaked in 0.85% NaCl. Cucumber seeds were sown in 10 cm-pots containing sterilized potting soil previously mixed with a P. putida BTP1 inoculum to a final concentration of 3.107 CFU/g or with an equal volume of sterile water for control plants. Cucumber were germinated at 25°C with a 16 h light photoperiod. Six and 12 days after sowing, 20 ml of a bacterial suspension at 108 CFU/ml were added as a drench to the roots. Control plants were watered with 20 ml of 0.85% NaCl. C. lagenarium infection Plants were placed in a humidity chamber 48 hours before infection experiments. The first true leaf was inoculated with 10 drops (10 μl) of a conidial suspension of C. lagenarium (106 spores/ml). In challenge experiments, inoculation was performed 5 (or 7) days after chemical treatment on the second or third leaf. Plant RNA extraction and analysis The extraction of total RNA from cucumber leaves (5 plants per treatment) was performed using a phenol/SDS method [52]. Northern hybridization of RNA fractionated by agarose-formaldehyde gel electrophoresis [52] was performed with a α-32P PR-8, CPR, CGT or CRG cDNA probe (Random Primed DNA labeling kit, Roche). mRNA Differential Display The mRNA differential display was performed using the differential display kit from Eurogentec (Belgium) and following the instructions of the supplier. PCR products were labeled with α-35S dATP, separated by electrophoresis on denaturing 6% polyacrylamide gels and visualized by autoradiography. PCR reactions showing differentially displayed bands were repeated twice on two different RNA samples in order to reduce the number of false-positives. Cloning and sequencing Differentially displayed bands were excised and eluted during 30 min in 100 μl of H2O followed by boiling during 10 min, then precipitated and suspended in 10 μl of H2O. A second PCR amplification was performed on 4 μl of DNA following the same protocol than for the differential display itself. Products were cloned in pGEM-T vector (Promega) and manually sequenced by the dideoxy sequencing method. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Slot-Blot hybridisation PCR amplified DNA from four to ten clones of each differentially displayed band was blotted on a positively charged nylon membrane using a filtration manifold and following the instructions of the supplier (Hoefer). Blots were hybridized with the 32P labeled RT-PCR corresponding products from either BTH or H2O treated plants. cDNA library construction and screening mRNA from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment) were purified with the PolyATractmRNA Isolation System III from Promega. The cDNA library was performed with the SMART cDNA Library Construction Kit from Clontech following the instructions of the supplier for cDNA synthesis by primer extension. The library was screened with 32P labeled cDNA probes. cDNA sequencing was performed at Genome Express, France. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Authors' contributions CB carried out the plants treatments, the molecular biology studies and the database research and drafted the manuscript. MO supplied the pathogen and participated in the challenge and ISR induction experiments. JD and PT participated in the design and coordination of the study. All authors read and approved the manuscript. Supplementary Material Additional File 1 PR-8 mRNA accumulation in cucumber leaves in response to different concentrations of BTH. Total RNA was extracted from cucumber leaves 24 h and 48 h after spraying with water, 10 μM BTH, 30 μM BTH, 100 μM BTH or from untreated leaves (UT). RNA was fractionated by electrophoresis on agarose gel. Northern blot was probed with α-32P labeled PR-8 cDNA. Loading of equal amounts of RNA was confirmed by ethidium bromide (Et Br) staining. Click here for file Acknowledgements This work was supported by a grant from the National Funds for Scientific Research (FNRS, Belgium) (Program FRFC n° 2.4.570.00). Figures and Tables Figure 1 Time course of PR-8 mRNA accumulation in cucumber leaves in response to chemical treatments. Total RNA was extracted from cucumber leaves at various time after spraying with water, 5 mM salicylic acid, 1 mM BTH, 50 mM L-α-amino butyric acid (ABA), 0.78 mM 2-thiouracil (2T-U) or from untreated leaves (UT). RNA (10 μg) was fractionated by electrophoresis on agarose gel. Northern blot was probed with α-32P labeled PR-8 cDNA (loading of equal amounts of RNA was confirmed by ethidium bromide staining). Figure 2 Local acquired resistance induced by 10 μM or 50 μM BTH. Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H2O, 10 μM BTH, 50 μM BTH or left untreated. The second true leaf was infected with C. lagenarium 5 days later. Plants were photographed 2 weeks postinoculation. Figure 3 Zoom in on an auto-radiogram of differentially displayed RT-PCR products. The RT-PCR were performed with primers 5'T11GA and 5'GATCTAACCG (lane 1 to 4), primers 5'T11GA and 5'GATCAATCGC (lane 5 to 8), primers 5'T11GA and 5'TACAACGAGG (lanes 9 to 12). Uneven lanes: H2O treated plants. Even lanes: 10 μM BTH treated plants. The arrows show bands corresponding to transcripts accumulating only or presenting a higher expression in BTH treated plants. Figure 4 Slot blotting of PCR amplified fragments identified in differential display experiments. PCR amplified DNA from four dd11 clones, nine dd6 clones and four dd5 clones respectively were hybridized with the 32P labeled RT-PCR corresponding products from either H2O or BTH treated plants. The arrows show DNA from clones containing differentially displayed fragments. Figure 5 Time course of mRNA accumulation in cucumber leaves after BTH treatment (A) or C. lagenarium inoculation (B). A: Total RNA was extracted from cucumber leaves at various time after application of 10 μM BTH or water (mock treatment). RNA (15 μg) from leaves 1 and 2 (treated) and leaves 3 and 4 (untreated) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with 32P-labeled PR-8, CPR and CGT cDNAs. B: Total RNA was extracted from cucumber leaves at various time after C. lagenarium inoculation. RNA (15 μg) from untreated leaves (UT), leaves 1 (infected) and leaves 2, 3 and 4 (non infected) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with 32P-labeled PR-8, CPR and CGT cDNAs. Equal loading of RNA was confirmed by ethidium bromide (Et Br) staining. A representative gel is shown. Table 1 Local acquired resistance induced by 10 μM or 50 μM BTH. untreated H2O 10 μM BTH 50 μM BTH Exp.1 66 ± 24 % 57 ± 25 % 12 ± 9 % 13 ± 11 % Exp.2 68 ± 28 % 71 ± 23 % 34 ± 30 % 5 ± 6 % Mean value 67 % 64 % 23 % 9 % Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H2O, 10 μM BTH, 50 μM BTH or left untreated. The second true leaf was infected with C. lagenarium 5 days later (ten infection sites per leaf). The percentages of spreading lesions (i.e. lesions showing a larger size than the initial inoculated area) observed 2 weeks after inoculation with C. lagenarium were calculated on the basis of 14 plants/treatment (experiment 1) and 16 plants/treatment (experiment 2) (means +/- standard deviations). Table 2 Systemic acquired resistance induced by 10 μM or 50 μM BTH. untreated H2O 10 μM BTH 50 μM BTH Exp.1 76 ± 20 % 69 ± 17 % 37 ± 19 % 18 ± 16 % Exp.2 96 ± 6 % 98 ± 3 % 39 ± 15 % 7 ± 9 % Mean value 86 % 83.5 % 38 % 12,5 % Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H2O, 10 μM BTH, 50 μM BTH or left untreated. The third true leaf was infected with C. lagenarium 7 days later (ten infection sites per leaf). The percentages of spreading lesions (i.e. lesions showing a larger size than the initial inoculated area) observed 2 weeks after inoculation with C. lagenarium were calculated on the basis of 18 plants/treatment (experiment 1) and 17 plants/treatment (experiment 2) (means +/- standard deviations). Table 3 Oligonucleotide primers used in RT-PCR that allowed to amplify the differentially displayed cDNA fragments. Clone name Downstream primer used Upstream primer used dd1 5' T11AA 5' CTGCTTGATG dd2 5' T11CG 5' GATCAATCGC dd3 5' T11CG 5' GATCAATCGC dd4 5' T11CG 5' TCGGTCATAG dd5 5' T11GA 5' AAACTCCGTC dd6 5' T11GA 5' TTTTGGCTCC dd7 5' T11GA 5' GATCTAACCG dd8 5' T11GA 5' GATCAATCGC dd9 5' T11GA 5' GATCAATCGC dd10 5' T11GA 5' TCGATACAGG dd11 5' T11GA 5' TACAACGAGG dd12 5' T11GG 5' TACAACGAGG Table 4 Characteristics of isolated cDNA clones. Clone name Clone length (bp) Accession N° Matching sequence from data base Origin of matching sequence and accession number Amino acid sequence % identity (*) dd3 774 AJ634908 Ras-related GTP-binding protein A. thaliana NP 177505 65 % (1e-73) dd4 1533 AJ635428 Translation releasing factor 2 A. thaliana NP 851097 70 % (7e-170) dd6 2192 AJ629867 β-1,4-N-acetylglucosaminyltranferase A. thaliana NP 172759 77 % (0.0) dd11 1588 AJ629866 Pathogenesis-related protein A. thaliana NP 565189 51 % (1e-51) * expected value of best match ==== Refs Heath MC Hypersensitive response related death Plant Mol Biol 2000 44 321 334 11199391 10.1023/A:1026592509060 Ward ER Uknes SJ Wlilliams SC Dincher SS Wiederhold DL Alexander DC Ahl-Goy P Métraux J-P Ryals JA Coordinate gene activity in response to agents that induce systemic acquired resistance Plant Cell 1991 3 1085 1094 12324583 10.1105/tpc.3.10.1085 Klessig DF Malamy J The salicylic acid signal in plants Plant Mol Biol 1994 26 1439 1458 7858199 Métraux J-P Signer H Ryals J Ward E Wyss-Benz M Gaudin J Rascdorf K Schmid E Blum W Inverardi B Increase in salicylic acid at the onset of systemic acquired resistance in cucumber Science 1990 250 1004 1006 Malamy J Carr JP Klessig DF Raskin I Salicylic acid : A likely endogenous signal in the resistance response of tobacco to viral infection Science 1990 250 1002 1004 Uknes S Winter AM Delaney T Vernooij B Morse A Friedrich L Nye G Potter S Ward E Ryals J Biological induction of systemic acquired resistance in Arabidopsis Mol Plant-Microbe Interact 1993 6 692 698 Delaney TP Uknes S Vernoij B Friedrich L Weymann K Negrotto D Gaffney T Gur-Rella M Kessmann H Ward E Ryals J A central role of salicylic acid in plant disease resistance Science 1994 266 1247 1250 Gaffney T Friedrich L Vernooij B Negrotto D Nye G Uknes S Ward E Kessmann H Ryals J Requirement of salicylic acid for the induction of systemic acquired resistance Science 1993 261 754 756 Cao H Bowling SA Gordon S Dong X Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance Plant Cell 1994 6 1583 1592 12244227 10.1105/tpc.6.11.1583 Delaney TP Friedrich L Ryals JA Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance Proc Natl Acad Sci U S A 1995 92 6602 6606 11607555 Després C DeLong C Glaze S Liu E Fobert PR The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors Plant Cell 2000 12 279 290 10662863 10.1105/tpc.12.2.279 Zhou J-M Trifa Y Silva H Pontier D Lam E Shah J Klessig DF NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid Mol Plant-Microbe Interact 2000 13 191 202 10659709 Fan W Dong X In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis Plant Cell 2002 14 1377 1389 12084833 10.1105/tpc.001628 Subramaniam R Desveaux D Spickler C Michnick SW Brisson N Direct visualization of protein interactions in plant cells Nat Biotechnol 2001 19 769 772 11479572 10.1038/90831 Spoel SH Koorneef A Claessens SMC Korzelius JP Van Pelt JA Mueller MJ Buchala AJ Métraux J-P Brown R Kazan K Van Loon LC Dong X Pieterse CMJ NPR1 modulates cross-talk between salicylate and jasmonate-dependent defense pathways through a novel fonction in the cytosol Plant Cell 2003 15 760 770 12615947 10.1105/tpc.009159 Glazebrook J Genes controlling expression of defense responses in Arabidopsis-2001 status Curr Opin Plant Biol 2001 4 301 308 11418339 10.1016/S1369-5266(00)00177-1 Glazebrook J Chen W Estes B Chang H-S Métraux J-P Zhu T Katagiri F Topology of the network integrating salicylate and jasmonate signal transduction derived from global expression phenotyping Plant J 2003 34 217 228 12694596 Kessmann H Staub T Hofmann C Maetzke T Herzog J Ward E Uknes S Ryals J Induction of systemic acquired resistance in plants by chemicals Annu Rev Phytopathol 1994 32 439 459 10.1146/annurev.py.32.090194.002255 Malamy J Sanchez-Casas P Henning J Guo A Klessig DF Dissection of the salicylic acid signaling pathway in tobacco Mol Plant-Microbe Interact 1996 9 474 482 Friedrich L Lawton K Ruess W Masner P Specker N Gut Rella M Meier B Dincher S Staub T Uknes S Métraux J-P Kessman H Ryals J A benzothiadiazole derivative induces systemic acquired resistance in tobacco Plant J 1996 10 61 70 10.1046/j.1365-313X.1996.10010061.x Görlach J Volrath S Knauf-Beiter G Hengy G Beckhove U Kogel K-H Oostendorp M Staub T Ward E Kessmann H Ryals J Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and disease resistance in wheat Plant Cell 1996 8 629 643 8624439 10.1105/tpc.8.4.629 Lawton KA Friedrich L Hunt M Weymann K Delaney T Kessmann H Staub T Ryals J Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway Plant J 1996 10 71 82 8758979 10.1046/j.1365-313X.1996.10010071.x Benhamou N Bélanger RR Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response Plant J 1998 14 13 21 15494051 10.1046/j.1365-313X.1998.00088.x Pieterse CMJ Van Loon LC Salicylic acid-independent plant defence pathways Trends Plant Sci 1999 4 52 58 10234273 10.1016/S1360-1385(98)01364-8 Kuc J Induced immunity to plant disease Bioscience 1982 32 854 860 Lawton KA Beck J Potter S Ward E Ryals J Regulation of cucumber class III chitinase gene expression Mol Plant-Microbe Interact 1994 7 48 57 8167370 Dalisay RF Kuc JA Persistence of reduced penetration by Colletotrichum lagenarium into cucumber leaves with induced systemic resistance and its relation to enhanced peroxidase and chitinase activities Physiol Mol Plant Pathol 1995 47 329 338 10.1006/pmpp.1995.1062 Narusaka Y Narusaka M Horio T Ishii H Comparison of local and systemic induction of acquired disease resistance in cucumber plants treated with benzothiadiazoles or salicylic acid Plant Cell Physiol 1999 40 388 395 10394634 Liang P Pardee AB Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction Science 1992 257 967 971 1354393 Métraux J-P Burkhart W Moyer M Dincher S Middlesteadt W Williams S Payne G Carnes M Ryals J Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysosyme / chitinase Proc Natl Acad Sci U S A 1989 86 896 900 2915985 Rasmussen JB Hammerschmidt R Zook MN Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv syringae Plant Physiol 1991 97 1342 1347 Jutidamrongphan W Mackinnon G Manners JM Scott KJ Sequence of a near-full length cDNA clone for a mRNA of barley induced by fungal infection Nucleic Acids Res 1989 17 9478 2587271 Ongena M Daayf F Jacques P Thonart P Benhamou N Paulitz TC Cornélis P Koedam N Bélanger RR Protection of cucumber against Pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and antibiosis Plant Pathol 1999 48 66 76 10.1046/j.1365-3059.1999.00315.x Irani K Goldschimidt-Clermont PJ Ras, superoxide and signal transduction Biochem Pharmacol 1998 55 1339 1346 10076523 10.1016/S0006-2952(97)00616-3 Ridley AJ The GTP-binding protein Rho Int J Biochem Cell Biol 1997 29 1225 1229 9451818 10.1016/S1357-2725(97)00052-6 Zhang S Klessig DF The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK Proc Natl Acad Sci 1998 95 7225 7230 9618567 10.1073/pnas.95.12.7225 Liu Y Zhang S Klessig DF Molecular cloning and characterization of a tobacco MAP kinase kinase that interacts with SIPK Mol Plant-Microbe Interact 2000 13 118 124 10656593 Ullah H Chen JG Young JC Im KH Sussman MR Jones AM Modulation of cell proliferation by heterodimeric G protein in Arabidopsis Science 2001 292 2066 2069 11408654 10.1126/science.1059040 Schiene K Puhler A Niehaus K Transgenic tobacco plants that express an antisense construct derived from a Medicago sativa cDNA encoding a Rac-related small GTP-binding protein fail to develop necrotic lesions upon elicitor infiltration Mol Gen Genet 2000 263 761 770 10905344 10.1007/s004380000248 Kawasaki T Henmi K Ono E Hatakeyama S Iwano M Satoh H Shimamoto K The small GTP-binding protein Rac is a regulator of cell death in plants Proc Natl Acad Sci U S A 1999 96 10922 10926 10485927 10.1073/pnas.96.19.10922 Sano H Ohashi Y Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants Proc Natl Acad Sci U S A 1995 92 4138 4144 11607540 Schultheiss H Dechert C Kogel KH Huckelhoven R A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley Plant Physiol 2002 128 1447 1454 11950993 10.1104/pp.010805 Ihara Y Yoshimura M Miyoshi E Nishikawa A Sultan AS Toyosawa S Ohnishi A Suzuki M Yamamura K-I Ijuhin N Taniguchi N Ectopic expression of N-acetylglucosaminyltransferase III in transgenic hepatocytes disrupts apolipoprotein B secretion and induces aberrant cellular morphology with lipid storage Proc Natl Acad Sci U S A 1998 95 2526 2530 9482919 10.1073/pnas.95.5.2526 Stanley P Biological consequences of overexpressing or eliminating N-acetylglucosaminyltransferase-TIII in the mouse Biochim Biophys Acta 2002 1573 363 368 12417419 10.1016/S0304-4165(02)00404-X Sato Y Takahashi M Shibukawa Y Jain SK Hamaoaka R Miyagawa J-I Yaginuma Y Honke K Ishikawa M Tanigushi N Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors J Biol Chem 2001 276 11956 11962 11134020 10.1074/jbc.M008551200 Shibukawa Y Takahashi M Laffont I Honke K Taniguchi N Down-regulation of hydrogen peroxide-induced PKCδ activation in N-acetylglucosaminyltransferase III-transfected HeLaS3 cells J Biol Chem 2003 278 3197 3203 12427758 10.1074/jbc.M207870200 Fitchette-Lainé AC Gomord V Cabanes M Michalski J-C Saint Macary M Foucher B Cavelier B Hawes C Lerouge P Faye L N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells Plant J 1997 12 1411 1417 9450345 10.1046/j.1365-313x.1997.12061411.x Wenderoth I von Schaewen A Isolation and characterization of plant N-acetylglucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants Plant Physiol 2000 123 1097 1108 10889259 10.1104/pp.123.3.1097 Strasser R Steinkellner H Boren M Altmann F Mach L Glossl J Mucha J Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana Glycoconj J 1999 16 787 791 11229321 10.1023/A:1007127815012 O'Donnell PJ Truesdale MR Calvert CM Dorans A Roberts MR Bowles DJ A novel tomato gene that rapidly responds to wound- and pathogen-related signals Plant J 1998 14 137 142 15494059 10.1046/j.1365-313X.1998.00110.x Pieterse CMJ van Wees SCM Hoffland E van Pelt JA van Loon LC Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression Plant Cell 1996 8 1225 1237 8776893 10.1105/tpc.8.8.1225 Ausubel FM Brent R Kingston RE Moore DD Seidman JG Smith JA Struhl K Current Protocols in molecular biology 1995 USA: Wiley Press

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-291533934010.1186/1471-2334-4-29Research ArticleHepatitis B virus variants in an HIV-HBV co-infected patient at different periods of antiretroviral treatment with and without lamivudine Santos Eneida A [email protected] Michel VF [email protected] Juçara [email protected] Selma A [email protected] Department of Virology, Oswaldo Cruz Institute, Rio de Janeiro, RJ 21045-900, Brazil2 Gaffrée and Guinle University Hospital, Rio de Janeiro, RJ 20270-004, Brazil2004 31 8 2004 4 29 29 3 6 2004 31 8 2004 Copyright © 2004 Santos et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Lamivudine inhibits replication of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and is commonly used as part of antiretroviral therapy. The main limitation in the use of lamivudine is resistant mutation selection. Most of these mutations affect the YMDD motif of the HBV DNA polymerase. The resistance occurs through M550V or M550I aminoacid replacements. The M550V variation may be accompanied by L526M mutation, notably in HIV-HBV co-infected patients. The aim of this study was to investigate mutations associated with lamivudine resistance in a hemodialysis patient chronically co-infected with HIV-1 and HBV, who was submitted to several antiretroviral treatments. Methods HBV isolates derived from three blood samples collected at different times of antiretroviral therapies with and without lamivudine, were titred and submitted to nucleotide sequencing. Results HBV isolate derived from a sample collected in 1999 during an antiretroviral treatment with lamivudine showed the lamivudine resistant double mutation (L526M, M550V). However, no mutation associated with lamivudine resistance was observed in the HBV genome derived from the sample collected during a period of treatment without lamivudine (2001). After reinstitution of lamivudine (2002), the predominant HBV population exhibited a rare triple mutation (V519L, L526M, M550V), which has previously been associated with an in vitro reduction of virus antigenicity (escape mutant). HBV DNA was detected at high levels (108–109 copies/ml) in the three blood samples. Conclusions Reintroduction of lamivudine as part of antiretroviral treatment in a patient who had developed lamivudine resistant HBV strains favored the predominance of an HBV isolate with reduced antigenicity. The absence of hepatitis acute exacerbation in this patient may be correlated to the absence of significant variations of the viral load, which was independent of the presence of mutations in the HBV DNA polymerase. ==== Body Background Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of transmission, mainly sexual, parenteral and vertical. Therefore, the prevalence of HBV serological markers is higher among HIV infected patients than in non-HIV infected individuals [1,2]. Considerable variations in the prevalence of HBV markers have been observed in HIV patients according to the geographical region and risk of exposure [3]. Recently, a prevalence of 68% of antibodies against hepatitis B core antigen (anti-HBc) was observed in HIV infected patients living in Rio de Janeiro, Brazil [4]. Co-infection with HIV interferes with the natural history of HBV infection and is associated with higher HBV DNA levels [5,6]. A more common progression to cirrhosis, despite a milder histological necro-inflammatory activity, has also been observed in cases of HIV-HBV co-infection [5]. Lamivudine is a nucleoside analogue that inhibits the reverse transcriptase activity of both HIV and HBV [7] and is commonly used in the treatment of both viral infections [8,9]. The HBV DNA loss due to lamivudine treatment is usually accompanied by significant histological and biochemical improvement [10]. The major limitation in the use of lamivudine is the selection of resistant mutations that may arise and accumulate during therapy. Most of these mutations usually affect the YMDD motif of the HBV DNA polymerase, by replacement of a methionine residue at position 550 with either valine (M550V) or isoleucine (M550I). Such mutations have notably been reported in HIV-HBV co-infected patients [11,12] who develop lamivudine resistance at an annual rate of 20%, with a projected rate of 90% after four years of therapy [13]. The consequences of drug resistance mutations for the evolution of HBV induced liver disease are currently under study. Hepatitis B acute exacerbation has been described after withdrawal of lamivudine therapy [14]. Such an exacerbation has also been associated with the appearance of YMDD mutants [14,15] and with a rapid increase of viral load [16]. The present study was performed to characterize HBV variants and genetic patterns of lamivudine resistant HBV strains in a patient co-infected with HIV-1 at different periods of an antiretroviral treatment with and without lamivudine. Methods Patient characteristics and serological markers A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 × 105 copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 105 copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003. DNA extraction and PCR assays for DNA sequencing Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously [17]. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5'-CCATATTCTTGGGAACAAGA-3', nt 2826-2845) and a mix of antisense primers S2 (5'-GGGTTTAAATGTATACCCAAAGA-3', nt 841-819) and S22 (5'-GTATTTAAATGGATACCCACAGA-3', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94°C, 30 s; 52°C, 1 min; 72°C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72°C. Amplification products (10 μL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light. Quantification of HBV DNA Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10-8 dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1–PS2, designed in the pre-S region and C1–C2 (core region), as described previously [4]. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction [18], equivalent to 104 HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 104 copies/ml by the last positive dilution. Molecular cloning and nucleotide sequencing Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed. Phylogenetic analysis Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows [19]. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree. Results Markers of HBV infection The patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999 (Figure 1). HIV treatment started in April 1996. Lamivudine was introduced as part of antiretroviral treatment in November 1996. HBV DNA was detected at high levels (108copies/ml) in the blood sample collected in March 1999, during this first period of lamivudine treatment (Figure 1). In May 1999, lamivudine was discontinued, and the sample analyzed during the period of lamivudine interruption (August 2001) showed the highest (109 copies/ml) HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (108copies/ml) after reintroduction of lamivudine (November 2002, Figure 1). HBV variants Nucleotide sequences of HBVs (pre-S/S region) derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A' (genotype A excluding A') and A' [20]. Recently, subgroups A-A' and A' were designated respectively as A1 and A2 [21] or Ae and Aa [22]. Isolates belonging to subgroup A' have been first identified in South Africa and circulate in a high proportion among HBV Brazilian isolates [23]. As can be observed on the phylogenetic tree represented in Figure 2, all clones belonged to subgroup A' and were closely related to each other. The six clones obtained during the two periods of lamivudine treatment (1999 and 2002) clustered separately (with a bootstrap value of 86%) from those obtained in 2001, during the period of lamivudine interruption. Table 1 shows the main amino acid changes observed in the S region of the genome. Two substitutions, H359Y in the polymerase and Y100C in the surface antigen (small S), were observed in all HBV sequences. These two changes were natural variation, characteristics of the HBV strain infecting the patient under study. All three clones derived from the sample collected in 1999, during the first lamivudine treatment, showed two lamivudine resistant mutations (L526M and M550V), also detected in the major viral population by direct sequencing. One clone (1-B57) showed an additional mutation related to drug resistance (V519L) that was not detected by direct sequencing. None of these three mutations associated with lamivudine resistance was observed in sequences derived from the second sample (2001). In contrast, all three clones derived from the third sample (2002) showed the three lamivudine resistant mutations. However, by direct sequencing of PCR products of this last sample, the electropherogram could detect two nucleotides (A and G) at the same sequence position indicating a mixture of V519 and L519 residues. Curiously, all HBV sequences obtained during the period without lamivudine treatment (2001) displayed a unique G473E substitution in the polymerase gene. Stop mutations were also observed in the small S protein, which was truncated at position 182 in clone 2-B14 and at position 216 in clone 2-B62 (Table 1). Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein (Table 1). Discussion Among lamivudine resistant mutations, those in the YMDD motif of the HBV DNA polymerase are the most common. The resistance occurs by replacement of a methionine residue at position 550 by either valine (M550V) or isoleucine (M550I). More rarely, HBV variant presenting M550S replacement may be selected during lamivudine treatment [24] The M550V variant may be accompanied by a mutation (leucine to methionine) at position 526 [25]. In the absence of HIV infection, the mutation at position 550 alone has been found in up to two-thirds of patients with lamivudine-resistant chronic hepatitis B. However, more than 90% of HIV-HBV co-infected patients display the double lamivudine resistant mutation at positions 526 and 550 [12]. Furthermore, the presence of L526M mutation in addition to mutation at position 550 seems to be associated with prolonged lamivudine treatment [26]. The double mutant has been shown to exhibit a 15-fold decrease of the in vitro susceptibility to lamivudine [27], since each mutation contributes to the loss of lamivudine sensitivity [28]. It is believed that the L526M mutation, when not accompanied with a mutation in the YMDD motif, does not confer lamivudine resistance [29]. Even so, the L526M mutation has been found alone in patients under lamivudine therapy [30]. Here, both L526M and M550V mutations were detected in all HBV sequences derived from the two blood samples collected during lamivudine treatment (1999 and 2002). Besides these common mutations, a third lamivudine resistant, rare mutation [11,31], namely V519L, was detected in one clone derived from the sample collected in 1999 as well as in all HBV sequences derived from the sample collected in 2002. This was in agreement with recent observations, showing that long lamivudine treatment may result in the predominance of this rare mutant [32]. The triple mutation V519L, L526M, M550V causes the concomitant amino acid substitutions E164D and I195M in the small S protein. It has been shown that this triple mutant has a reduced in vitro affinity to anti-HBs antibodies, similar to the hepatitis B vaccine escape mutant G145R [11,31]. The accumulation of mutations in the HBV genome should be monitored in order to guide patient management adequately. There is a general consensus that the lamivudine resistant single mutants in the YMDD motif (M550V/I) replicate substantially more slowly than the wild type. The addition of the mutation at position 526 may act as a compensatory change that partially restores the replication fitness of the virus [33]. Here, in agreement with this observation, HBV DNA was detected at high levels (about 108 copies/ml) in the blood sample collected in 1999, in which the double mutant represents the major viral population. Similar high HBV loads were observed in the sample containing the triple mutant (2002). This result is in agreement with a recent study, showing that V519L mutation also enhances viral replication [34]. The HBV population that emerged during the interruption of lamivudine treatment did not show mutations at polymerase positions 519, 526 and 550. However, another substitution, namely G473E, was observed that was associated to G119R substitution in the small S protein. Such a virus might be present as a minor population but not detected during the first lamivudine treatment. Although two out three clones of this population possessed stop codon mutations in S gene, HBV load (109 copies/ml) was moderately higher during the lamivudine interruption period than that found during lamivudine periods. HBV chronically infected patients submitted to lamivudine treatment may have acute exacerbation after withdrawal of drug therapy or when lamivudine resistance emerges [8,14-16,26,35-37]. In HIV-HBV co-infected patients, withdrawal of lamivudine may result in severe [8,35,37] or fulminant [37] hepatitis. Factors that trigger severe hepatitis in these cases are not well known. Both viral load and genome variations have been implicated in the pathogenesis of acute exacerbation. Here, the patient under study did not present clinical signs of HBV infection. The absence of acute exacerbation after the emergence of lamivudine resistant variants may be correlated to the absence of significant variations of the viral load. Indeed, HBV DNA was detected at high levels (108–109 copies/ml) at different periods of antiretroviral treatment with and without lamivudine. Conclusions A rare HBV triple mutant, belonging to genotype A, subgroup A', appeared predominantly in a patient submitted to lamivudine as part of HIV treatment. This type of mutant, previously found in isolates belonging to genotypes A, D and G [15,36], may behave as a vaccine escape mutant. Understanding the circumstances leading to the appearance of such HBV strains may help to guide future therapies in HIV-HBV co-infected patients. A correlation may exist between acute exacerbation of hepatitis B and HBV load in lamivudine treated patients. Competing interests None declared. Authors' contributions EAS and MVS carried out cloning and sequencing of HBV DNA. JA was involved in clinical evaluation of the patient and supervised the antiretroviral treatment. SAG conceived the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The authors acknowledge Drs Christian Niel and Regina MB Martins for the critical reading of the manuscript. This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Figures and Tables Figure 1 HBV load (vertical bars) and ALT levels (curve) during antiretroviral therapies. Figure 2 Phylogenetic tree of HBV isolates constructed with the neighbor-joining method, and based on the nucleotide sequences of the entire pre-S/S region (nt positions 2854 to 833). Isolates whose names begin by 1,2,3 were molecular clones from this study derived from samples collected in 1999, 2001, and 2002, respectively. The other isolates are designated by their GenBank accession numbers. Genotype A isolates clustered in two subgroups, designated A-A' (genotype A excluding A') and A' [27]. The sequence AY090458 belongs to genotype F and was used as an outgroup. Numbers at internal nodes indicate percentage of 100 bootstrap replicates that support the branch. Only values > 85% are indicated. The horizontal bar provides a genetic distance. Table 1 Amino acid replacements in polymerase and surface antigen (small S) Year of collect Lamivudine HBV sequences Polymerase residues S residues 359 473 519 526 550 100 119 164 182 195 216 1999 Yes Direct Y G V M V C G E W M L 1-B40 Y G V M V C G E W M L 1-B52 Y G V M V C G E W M L 1-B57 Y G L M V C G D W M L 2001 No Direct Y E V L M C R E W I L 2-B14 Y E V L M C R E * I L 2-B62 Y E V L M C R E W I * 2-B63 Y E V L M C R E W I L 2002 Yes Direct Y G L/V M V C G D/E W M L 3-B02 Y G L M V C G D W M L 3-B06 Y G L M V C G D W M L 3-B71 Y G L M V C G D W M L Consensus of genotype A H G V L M Y G E W I L Lamivudine resistant mutations in the viral DNA polymerase and their counterparts in the small surface antigen are marked in bold. Asterisks represent stop mutations. ==== Refs Enzensberger R Braun W July C Helm EB Doerr HW Prevalence of antibodies to human herpesviruses and hepatitis B virus in patients at different stages of human immunodeficiency virus (HIV) infection Infection 1991 19 140 145 1653770 Saillour F Dabis F Dupon M Lacoste D Trimoulet P Monlun E Ragnaud JM Morlat P Pellegrin JL Fleury H Couxigou P Prevalence and determinants of antibodies to hepatitis C virus and markers for hepatitis B virus infection in patients with HIV infection in Aquitaine. Groupe d'Epidemiologie Clinique du SIDA en Aquitaine BMJ 1996 313 461 464 8776313 Twu SJ Detels R Nelson K Visscher BR Kaslow R Palenicek J Relationship of hepatitis B virus infection to human immunodeficiency virus type 1 infection J Infect Dis 1993 167 299 304 8421164 Santos EA Yoshida CFT Rolla VC Mendes JM Vieira IF Arabe J Gomes SA Frequent occult hepatitis B virus infection in patients infected with human immunodeficiency virus type 1 Eur J Clin Microbiol Infect Dis 2003 22 92 98 12627282 Colin JF Cazals-Hatem D Loriot MA Martinot-Peignoux M Pham BN Auperin A Degott C Benhamou JP Erlinger S Valla D Marcellin P Influence of human immunodeficiency virus infection on chronic hepatitis B in homosexual men Hepatology 1999 29 1306 1310 10094979 10.1002/hep.510290447 Gilson RJ Hawkins AE Beecham MR Ross E Waite J Briggs M Interactions between HIV and hepatitis B virus in homosexual men: effects on the natural history of infection AIDS 1997 11 597 606 9108941 10.1097/00002030-199705000-00007 Benhamou Y Dohin E Lunel-Fabiani F Poynard T Huraux JM Katlama C Opolon P Gentilini M Efficacy of lamivudine on replication of hepatitis B virus in HIV-infected patients Lancet 1995 345 396 397 7845151 10.1016/S0140-6736(95)90388-7 Bessesen M Ives D Condreay L Lawrence S Sherman KE Chronic active hepatitis B exacerbations in human immunodeficiency virus-infected patients following development of resistance to or withdrawal of lamivudine Clin Infect Dis 1999 28 1032 1035 10452630 Lai CL Yuen MF Profound suppression of hepatitis B virus replication with lamivudine J Med Virol 2000 61 367 373 10861648 10.1002/1096-9071(200007)61:3<367::AID-JMV15>3.0.CO;2-A Honkoop P de Man RA Zondervan PE Schalm SW Histological improvement in patients with chronic hepatitis B virus infection treated with lamivudine Liver 1997 17 103 106 9138281 Cooley L Ayres A Bartholomeusz A Lewin S Crowe S Mijch A Locarnini S Sasadeusz J Prevalence and characterization of lamivudine-resistant hepatitis B virus mutations in HIV-HBV co-infected individuals AIDS 2003 17 1649 1657 12853747 10.1097/00002030-200307250-00009 Thibault V Benhamou Y Seguret C Bochet M Katlama C Bricaire F Opolon P Poynard T Agut H Hepatitis B virus (HBV) mutations associated with resistance to lamivudine in patients coinfected with HBV and human immunodeficiency virus J Clin Microbiol 1999 37 3013 3016 10449493 Leung NW Lai CL Chang TT Guan R Lee CM Ng KY Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: results after 3 years of therapy Hepatology 2001 33 1527 1532 11391543 10.1053/jhep.2001.25084 Honkoop P de Man RA Niesters HG Zondervan PE Schalm SW Acute exacerbation of chronic hepatitis B virus infection after withdrawal of lamivudine therapy Hepatology 2000 32 635 639 10960461 10.1053/jhep.2000.16333 Liaw YF Chien RN Yeh CT Tsai SL Chu CM Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy Hepatology 1999 30 567 572 10421670 10.1002/hep.510300221 Liu CJ Chen PJ Lai MY Kao JH Chen DS Hepatitis B virus variants in patients receiving lamivudine treatment with breakthrough hepatitis evaluated by serial viral loads and full-length viral sequences Hepatology 2001 34 583 589 11526546 10.1053/jhep.2001.27220 Niel C Moraes MT Gaspar AM Yoshida CF Gomes SA Genetic diversity of hepatitis B virus strains isolated in Rio de Janeiro Brazil J Med Virol 1994 44 180 186 7852959 Gomes SA Yoshida CF Niel C Detection of hepatitis B virus DNA in hepatitis B surface antigen-negative serum by polymerase chain reaction: evaluation of different primer pairs and conditions Acta Virol 1996 40 133 138 8891092 Van de Peer Y de Wachter R TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment Comput Applic Biosci 1994 10 569 570 Bowyer SM van Staden L Kew MC Sim JG A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa J Gen Virol 1997 78 1719 1729 9225049 Kimbi GC Kramvis A Kew MC Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa J Gen Virol 2004 85 1211 1220 15105537 10.1099/vir.0.19749-0 Sugauchi F Kumada H Acharya SA Shrestha SM Gamutan MT Khan M Gish RG Tanaka Y Kato T Orito E Ueda R Miyakawa Y Mizokami M Epidemiological and sequence differences between two subtypes (Ae and Aa) of hepatitis B virus genotype A J Gen Virol 2004 85 811 820 15039524 10.1099/vir.0.79811-0 Araujo NM Mello FC Yoshida CF Niel C Gomes SA High proportion of subgroup A' (genotype A) among Brazilian isolates of Hepatitis B virus Arch Virol 2004 149 1383 1395 15221538 10.1007/s00705-003-0269-4 Niesters HG De Man RA Pas SD Fries E Osterhaus AD Identification of a new variant in the YMDD motif of the hepatitis B virus polymerase gene selected during lamivudine therapy J Med Microbiol 2002 51 695 699 12171302 Bartholomew MM Jansen RW Jeffers LJ Reddy KR Johnson LC Bunzendahl H Condreay LD Tzakis AG Schiff ER Brown NA Brown NA. Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation Lancet 1997 349 20 22 8988118 10.1016/S0140-6736(96)02266-0 Yeh CT Chien RN Chu CM Liaw YF Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy Hepatology 2000 31 1318 1326 10827158 10.1053/jhep.2000.7296 Xiong X Flores C Yang H Toole JJ Gibbs CS Mutations in hepatitis B DNA polymerase associated with resistance to lamivudine do not confer resistance to adefovir in vitro Hepatology 1998 28 669 673 10.1002/hep.510280629 Allen MI Deslauriers M Andrews CW Tipples GA Walters KA Tyrrell DL Brown N Condreay LD Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group Hepatology 1998 27 1670 1677 9620341 10.1002/hep.510270628 Malik AH Lee WM Hepatitis B therapy: the plot thickens Hepatology 1999 30 579 581 10421673 10.1002/hep.510300238 Yuen MF Sablon E Hui CK Yuan HJ Decraemer H Lai CL Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy Hepatology 2001 34 785 791 11584376 10.1053/jhep.2001.27563 Torresi J Earnest-Silveira L Deliyannis G Edgtton K Zhuang H Locarnini SA Fyfe J Sozzi T Jackson DC Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy Virology 2002 293 305 313 11886250 10.1006/viro.2001.1246 Roque-Afonso MF Ferey MP Mackiewicz V Fki L Dussaix E Monitoring the emergence of hepatits B virus polymerase gene veriants during lamivudine therapy in human immunodeficiency virus coinfeted patients: performance of CLIP sequencing and line probe assay Antivir Ther 2003 8 627 634 14760897 Ono SK Kato N Shiratori Y Kato J Goto T Schinazi RF Carrilho FJ Omata M The polymerase L528M mutation cooperates with nucleotide binding-site mutations increasing hepatitis B virus replication and drug resistance J Clin Invest 2001 107 449 455 11181644 Delaney WE 4thYang H Westland CE Das K Arnold E Gibbs CS Miller MD Xiong S The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro J Virol 2003 77 11833 11841 14557667 10.1128/JVI.77.21.11833-11841.2003 Neau D Schvoerer E Robert D Dubois F Dutronc H Fleury HJ Ragnaud JM Hepatitis B exacerbation with a precore mutant virus following withdrawal of lamivudine in a human immunodeficiency virus-infected patient J Infect 2000 41 192 194 11023772 10.1053/jinf.2000.0724 Thabut D Thibault V Benhamou Y Bernard B Aubron-Olivier C Poynard T Di Martino V Successful control of subfulminant hepatitis related to lamivudine-resistant hepatitis B virus in an HIV-infected patient AIDS 2001 15 2463 2464 11774835 10.1097/00002030-200112070-00020 Bonacini M Kurz A Locarnini S Ayres A Gibbs C Fulminant hepatitis B due to a lamivudine-resistant mutant of HBV in a patient coinfected with HIV Gastroenterology 2002 122 244 245 11806370

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-601534502710.1186/1471-2407-4-60Research ArticleSurvival of patients with metastatic breast cancer: twenty-year data from two SEER registries Tai Patricia [email protected] Edward [email protected] Vincent [email protected] Gábor [email protected] Georges [email protected] University of Saskatchewan, Faculty of Medicine, Saskatoon; Department of Radiation Oncology, Regina, Canada2 Radiation Oncology Program, London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada3 AZ-VUB, Oncologisch Centrum, Jette, Belgium4 Bács-Kiskun County Teaching Hospital, Surgical Pathology, Kecskemét, Hungary5 Geneva University Hospitals, Department of Gynecology and Obstetrics, gynecologic oncology and senology, Geneva, Switzerland2004 2 9 2004 4 60 60 22 4 2004 2 9 2004 Copyright © 2004 Tai et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Many researchers are interested to know if there are any improvements in recent treatment results for metastatic breast cancer in the community, especially for 10- or 15-year survival. Methods Between 1981 and 1985, 782 and 580 female patients with metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries of the Surveillance, Epidemiology, and End Results (SEER) database. The lognormal statistical method to estimate survival was retrospectively validated since the 15-year cause-specific survival rates could be calculated using the standard life-table actuarial method. Estimated rates were compared to the actuarial data available in 2000. Between 1991 and 1995, further 752 and 632 female patients with metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries. The data were analyzed to estimate the 15-year cause-specific survival rates before the year 2005. Results The 5-year period (1981–1985) was chosen, and patients were followed as a cohort for an additional 3 years. The estimated 15-year cause-specific survival rates were 7.1% (95% confidence interval, CI, 1.8–12.4) and 9.1% (95% CI, 3.8–14.4) by the lognormal model for the two registries of Connecticut and San Francisco-Oakland respectively. Since the SEER database provides follow-up information to the end of the year 2000, actuarial calculation can be performed to confirm (validate) the estimation. The Kaplan-Meier calculation for the 15-year cause-specific survival rates were 8.3% (95% CI, 5.8–10.8) and 7.0% (95% CI, 4.3–9.7) respectively. Using the 1991–1995 5-year period cohort and followed for an additional 3 years, the 15-year cause-specific survival rates were estimated to be 9.1% (95% CI, 3.8–14.4) and 14.7% (95% CI, 9.8–19.6) for the two registries of Connecticut and San Francisco-Oakland respectively. Conclusions For the period 1981–1985, the 15-year cause-specific survival for the Connecticut and the San Francisco-Oakland registries were comparable. For the period 1991–1995, there was not much change in survival for the Connecticut registry patients, but there was an improvement in survival for the San Francisco-Oakland registry patients. ==== Body Background Prospective trials have the disadvantages of requiring a long time to complete, and using highly selected patient subgroups in tertiary centers. While one waits for the results to mature, this delays additional research to improve treatment. If there were a method that allowed earlier prediction of the results of prospective trials, advances in cancer treatment could be attained within a shorter time period. There is a parametric lognormal model, proposed by Boag [1-3] that had been retrospectively validated in the literature, and could be used prospectively for clinical trials to predict long-term survival rates several years earlier than would otherwise be possible using the standard life-table/actuarial Kaplan-Meier method of calculation [4]. The prognosis for metastatic breast cancer is generally poor and therefore it is believed that statistical prediction models for long-term survival rates are not needed. Nevertheless, specific subgroups of metastatic breast cancer patients exist, for which depending on the treatment given, the prognosis is improved so that some patients can survive for some time, particularly for those with limited organs involvement such as involvement with bone and/or skin only. In this situation, for which the present study was relevant, a prediction model, even for metastatic breast cancer, can be useful. Breast cancer, among other cancers, has the highest incidence in women, and many studies are currently in progress to assess treatment regimens. If, even for a subgroup of patients, the 10- and 15-year survival rates can be predicted from follow-up data available only 3 years after a 5-year diagnosis period, this would be a useful means of obtaining study results earlier than would otherwise have been possible. For example, a 15-year survival rate calculated by the Kaplan-Meier method requires at least some patients to have been followed for 15 years. In addition prediction model such as the lognormal model can also be used to review the progress of treatment results for a specific period from a treatment center, and to compare that with another specific period of the same treatment center to evaluate the potential impact for any possible change in treatment policy or guideline. Boag's lognormal model for long-term cancer survival rates has been available for use for some 50 years. When the lognormal model was first proposed in the 1940s, it was difficult to implement because of a lack of computing power, and lack of good quality long-term follow-up data from cancer registries. Since 1970s the model has been used by some authors in breast cancer, cervix uteri cancer, head and neck cancer, intraocular melanoma, choroidal-ciliary body melanoma, and small cell lung cancer [5-10]. Currently, although the computing power is sufficient, good quality follow-up data on a sufficient number of patients are seldom available, and it can be a limitation for its application. Large data registry such as the Surveillance, Epidemiology, and End Results (SEER) data [11] with good long-term follow-up data available can overcome this potential limitation. Methods Between 1981 and 1985, 782 and 580 female patients of metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries from the SEER database using SEER*Stat 5.0 software. The two registries were chosen because they are two of the earliest registries, with a large population. The data used were survival time, vital status, cause of death, age at diagnosis, and race. The cause-specific survival was defined as the interval from the date of diagnosis to the date of death from breast cancer or the last follow-up date for censoring purposes, if the patient was alive and still being followed at the time of analysis. The survival time of the uncured group of patients who died of breast cancer had been verified to follow a lognormal distribution previously [12]. Next, between 1991 and 1995, 752 and 632 female patients of metastatic breast cancer were extracted respectively from the two registries. The data were used to estimate the 15-year cause-specific survival rates before the year 2005. To be comparable, for both the 1981–1985 and 1991–1995 cohorts, the staging system used was the SEER historical system (classified as localized, regional, or distant, based on combined pathologic and clinical data). The choice of 1981–1985 and 1991–1995 has the advantage that the two time periods are not too far apart otherwise there would be too much changes of medical practice. These time periods have a minimum of 5 years follow-up. The overall survival rates (OSR) of the two time periods were calculated using the Kaplan-Meier method. The actual relative survival rates (RSR) were calculated using SEER*Stat 5.0 software. The modified version of period analysis [13] was applied using the Hakulinen method [14] to obtain more up-to-date absolute survival rates (ASR) and relative survival rates (RSR) for comparison purpose with a computer program run by Microsoft Excel software. Validation of the lognormal model The validation of the lognormal model has two phases. Phase 1 tests the goodness of fit to a lognormal distribution of the survival time of those cancer patients who died with their disease present, termed an uncured group with a fraction of 1-C, where C is the cured proportion of patients. The lognormal distribution is similar to the normal distribution in that if the variable in the normal is time t, the variable in the lognormal is the logarithm of t. In other words, the investigators attempt to show that the logarithm of the survival time follows a normal distribution. Phase 2 attempts to show that the lognormal model, using short-term follow-up data, can predict long-term survival rates comparable to those calculated by the Kaplan-Meier life-table method with long-term available. This model can be used to estimate long-term cause-specific survival rates (CSSR) by a maximum likelihood method (e.g., 10-year and 15-year survival rates) from only short-term follow-up data. The maximum likelihood method is used to estimate the CSSR at time τ, and is calculated as [C+(1-C)·Q]·100%, where Q is the integral of the lognormal distribution between the limits time τ and infinity. The lognormal statistical model had been validated in stages III and IV breast cancer in a previous publication that survival rates could be estimated several years earlier than is possible using the standard life-table actuarial method [12]. The survival time of unsuccessfully treated cases could be represented by a lognormal distribution, the long-term survival rates were predicted by Boag's method using a computer program run by Microsoft Excel. In this parametric lognormal model, the standard deviation S was fixed, and only the two remaining parameters, mean M and proportion cured C, were kept floating when using the maximum likelihood method. Multiple iterations converged to a stable solution for C. A 5-year period of diagnosis could be selected and patients followed as a cohort for an additional 3 years. The current study was for metastatic breast cancer patients treated between 1981 and 1985, with follow-up to the end of year 2000, making the series ideal for validating purposes. For example, for cases diagnosed during the 5-year period, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1988 (i.e., 3 years after 1985). The 15-year survival rate prediction was then validated by Kaplan-Meier life-table calculations using the follow-up data available in 2000. For metastatic breast cancer patients treated between 1991 and 1995, and follow-up to the end of year 2000, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1998 (i.e., 3 years after 1995) before the year 2005. Results From the cohort of 1981–1985 inclusively, 782 patients from the Connecticut registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 7.1% (95% CI, 1.8–12.4). The 15-year CSSR was 8.3% (95% CI, 5.8–10.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. From the cohort of 1981–1985 inclusively, 580 patients from the San Francisco-Oakland registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 9.2% (95% CI, 3.9–14.5). The 15-year CSSR was 7.0% (95% CI, 4.3–9.7) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. Using the same method, the cohort of 1991–1995 inclusively, 752 patients from the Connecticut registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 12.6% (95% CI, 7.3–17.9). The 10-year CSSR was 11.3% (95% CI, 7.8–14.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 9.1% (95% CI, 3.8–14.4), which cannot be validated before 2005. For the cohort of 1991–1995 inclusively, 632 patients from the San Francisco-Oakland registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 17.0% (95% CI, 12.1–21.9). The 10-year CSSR was 15.9% (95% CI, 11.4–20.4) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 14.7% (95% CI, 9.8–19.6), which cannot be validated before 2005. For the period 1991–1995, there was not much change of only about 2% absolute percentage point in the predicted 15-year CSSR for the Connecticut registry, but there was an improvement of about 6% absolute percentage points for the San Francisco-Oakland registry when compared with the period 1981–1985 15-year CSSR, which was validated by the Kaplan-Meier calculation. (Table 1) For comparison purpose, the actual OSR and RSR were compared with the ASR and RSR obtained by the period analysis. (Tables 2 and 3) It was found that there were more patient survival improvements shown in the actual OSR and RSR for the San Francisco-Oakland registry, but not much for the Connecticut registry. However the period analysis results did not show such improvements. Discussion Lognormal model Rutqvist studied the fit of Boag's lognormal model to the survival times of 8170 breast cancer cases reported to the Swedish Cancer Registry during 1961–1963. The model fitted the 1961–1963 data well for the entire case material and for patients aged less than 70 years. In this registry, the lognormal model did not fit the data for patients aged greater than 70 years, who were more likely to be censored because of coincidental causes of death. Another disadvantage stated by the author was that large number of patients was required to obtain estimates with reasonably small standard errors for breast cancer. With another series of the Norwegian Cancer Registry of 14,000 breast cancer cases, Rutqvist et al. [15] deduced that lognormal is the best model because other models did not fit the observed survival in all stages, ages, and time periods (two-parameter models, such as exponential or extrapolated actuarial, or three-parameter models, such as sum of two exponential, exponential with shoulder, Weibull). Both the exponential and extrapolated actuarial models assume that the conditional relative survival is lowest immediately after treatment. With the lognormal model, the survival curve has a low initial mortality that rapidly increases to a maximum, with a slow decrease in the mortality after the maximum has occurred. Requirements for using the lognormal model The lognormal model can only predict cause-specific survival, because other coincidental causes of death are too unpredictable (e.g., the rate of stroke). Therefore, overall survival cannot be predicted. The maximum likelihood method is the most accurate method for fitting the lognormal model with the smallest mean squared error. However, there are some requirements for its use. The maximum likelihood method fails to converge to a stable solution using the initial estimates if there is extensive censoring within the data. This occurs if patients are lost to follow-up or die from coincidental non-cancerous causes. The frequency of failure to yield a successful fit for lognormality was greater when one-fourth of cases were designated as lost to follow-up. Gamel et al. established a stable linear algorithm for fitting the lognormal model to survival data. To achieve convergence, some authors have fixed one or two parameters of the lognormal model to pre-selected values to simplify the iterative procedure required for convergence [6]. Some prognostic factors follow lognormal distribution Prognostic factors in patients with distant metastases at the time of diagnosis were investigated by Rudan et al. [16], and Chapman et al. [17], primary tumor size was a significant prognostic factor. Engel et al. [18] found that the number of metastatic cases and the time to metastasis depended on the tumor diameter at diagnosis. Cell growth is essential for the development of tumors. Tumor size is therefore the most important factor in describing tumor biology. As the tumor size increases, the probability of node-positivity increases. Another study group also found this correlation up to 5 cm [19]. Tubiana and Koscielny [20] have found a highly significant correlation between tumor size and the probability of distant metastasis. The distribution of tumor sizes at metastatic spread was lognormal with a median diameter equal to 3.5 cm. The patients were subdivided into 3 groups according to the histological grade. In each subgroup there was a significant correlation between tumor size and the probability of distant spread. The distributions were lognormal and the median size was markedly larger for grade 1 tumors. A number of quantitative postmortem observations regarding the size distribution of metastases have been published [21-23]. These studies revealed a skewed distribution with a high proportion of smaller metastases, and a significant tail extending to the larger metastases, consistent with a lognormal distribution. The more detailed measurements from human liver metastases provided by Yamanami et al. were found to approximate the lognormal distribution reasonably well. A hypothesis was proposed by Kendal [24] that the time available for the growth of metastases is normally distributed, presumably as a consequence of the summation of multiple independently distributed time intervals from each of the steps and of the Central Limit Theorem. For exponentially growing metastases, the corresponding size distribution would be lognormal; Gompertzian growth would imply a modified (Gompertz-normal) distribution, where larger metastases would occur less frequently as a consequence of a decreased growth rate. These two size distributions were evaluated against 18 human autopsy cases where precise size measurements had been collected from over 3900 macroscopic hematogenous organ metastases. The lognormal distribution provided an approximate agreement. Its main deficiency was a tendency to over-represent metastases greater than 10 mm diameter. These observations supported the hypothesis of normally distributed growth times, and qualified the utility of the lognormal and Gompertz-normal distributions for the size distribution of metastases. Why is the lognormal model applicable to so many organ sites [3,6-10,12,25-36] (Table 4)? Boag's explanation for the lognormal survival time distribution was that if the patient was not cured by treatment, the length of the remaining survival time would be dependent principally on the growth rate of the tumor remnants. Pearlman [37] estimated the growth rates of breast cancer that recurred in the scar, assuming that the recurrence started from a single cell. He found that the growth rates were approximately lognormally distributed. Likewise, von Fournier et al. [38] found that the growth rates of breast cancers followed by serial mammography were lognormally distributed. Variation of survival rates over time In order to determine whether current programs for the management of metastatic breast cancer have led to improved patient survival, Debonis et al. [39] determined the median survival times for five-year intervals of 849 patients admitted to the City of Hope National Medical Center with metastatic breast cancer from 1955 to 1980. Survival times in each of the clinical subsets remained unchanged during the period of observation, regardless of the therapeutic modalities included in the treatment regimens. The study indicates that changes in palliative therapy for metastatic breast cancer during the 25 years of observation have not influenced overall survival. On the contrary, Dickman et al. [40] studied the survival of cancer patients in Finland during the years 1955–1994. The 5-year RSR for distant metastases breast cancer had increased from 10% for the period 1955–1964 to 22% for 1985–1994. The tumor registry at Yale-New Haven Hospital, which began recording data in 1920, was utilized by Todd et al. [41] to examine the ultimate outcome of all breast cancer patients who were initially diagnosed at Yale with metastatic breast cancer. The median survival of these patients increased steadily from 21 months in 1920 to 41 months in the decade from 1970 to 1980. The percentage of women actually surviving 5 years increased from 5% in the 1920s to approximately 25% in the 1960s. Despite the use of combination drug programs in the 1970s, the percentage of these patients remaining alive at 5 years remained near 25%. Firm conclusions cannot be made from a retrospective study spanning 60 years, although the trends depicted the lack of continued improvement indicate that the current therapeutic approach to metastatic breast cancer in that period may not result in dramatic improvement in overall survival. Geographical variation of survival rates Farrow et al. [42] documented substantial geographical variation in patterns of treatment of cancer and other diseases. Because cancer treatment is not uniform nationwide in the States, survival following the diagnosis of cancer might also be expected to vary geographically. Survival data from the nine population-based registries in the SEER Program were analyzed for cancers of the stomach, colon, rectum, lung, breast, uterus, ovary, prostate, and bladder. The patients included all non-Hispanic white patients diagnosed with cancer of one of the selected sites during 1983–1991. Regional variation in crude five-year survival rates across the nine SEER areas was most marked for cancers of the uterus and prostate. For uterine cancer, for example, five-year survival ranged from 73.2% in Connecticut to 84.0% in Hawaii. Less marked variation was observed for cancers of the colon, rectum, and breast. For cancers of the bladder, ovary, stomach, and lung, survival rates five years after diagnosis were relatively invariant across the SEER areas. Maggard et al. [43] also found that variations in the breast cancer mortality rates exist between states. A nearly 50% increase is observed between the states with the highest and lowest mortality rates. Adjusted analyses demonstrated that stage at presentation is a more important predictor of mortality variation than treatment differences. Goodwin et al. [44] examined breast cancer incidence, survival, and mortality in the 66 health service areas covered by the SEER program for women aged 65 and older at diagnosis. They found that there was considerable geographic variation in survival from breast cancer among older women, and this contributed to variation in breast cancer mortality. The elevated mortality in the Northeast is apparent only in older women [45]. For women aged 65 years and older, breast cancer mortality is 26% higher in New England than in the South, while incidence is only 3% higher. Breast cancer mortality for older women by state correlates poorly with incidence (r = 0.28). The above-mentioned results are consistent with that from the present study: the Connecticut registry has lower CSSR than the San Francisco-Oakland registry for the period 1991–1995. The Connecticut cohort has median age at diagnosis of 66 (range 25–103), while the San Francisco-Oakland cohort has lower median age of 63 (range 26–96). It could be argued that new treatments evolved in the recent decade have improved the survival of the breast cancer patients, and younger patients benefit more than the older patients. Apart from treatment offered, changes of survival rates over time or geographical areas can be due to co-morbidities or other characteristics such as race, age, and differences in staging procedures. Conclusions For the period 1981–1985, the 15-year cause-specific survival for the Connecticut and the San Francisco-Oakland registries were comparable. For the period 1991–1995, there was not much change in survival for the Connecticut registry patients, but there was an improvement in survival for the San Francisco-Oakland registry patients. List of abbreviations CSSR: Cause-specific survival. SEER: Surveillance, Epidemiology, and End Results. OSR: Overall survival rate. ASR: Absolute survival rate. RSR: Relative survival rate. Competing interests None declared. Authors contributions PT: Data analysis and writing of manuscript. EY, VVH, GC, GV: Critical appraisal of manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments PT: Saskatchewan Cancer Agency Research Grant Award 2792. GC: János Bolyai Research Fellowship from the Hungarian Academy of Sciences. Figures and Tables Table 1 10- and 15-year cause-specific survival rates for Connecticut and San Francisco-Oakland registries for 1981–1985 and 1991–1995 cohorts calculated by lognormal model (LN) and Kaplan-Meier (KM) method with 95% confidence intervals in brackets Connecticut San Francisco-Oakland Year 1981–1985 1991–1995 1981–1985 1991–1995 LN KM LN KM LN KM LN KM 10 11.5 (6.2–16.8) 11.0 (8.5–13.5) 12.6 (7.3–17.9) 11.3 (7.8–14.8) 12.3 (7.0–17.6) 9.7(6.8–12.6) 17.0 (12.1–21.9) 15.9 (11.4–20.4) 15 7.1 (1.8–12.4) 8.3 (5.8–10.8) 9.1 (3.8–14.4) N.A. 9.2 (3.9–14.5) 7.0 (4.3–9.7) 14.7 (9.8–19.6) N.A. N.A. = not available Table 2 10- and 15-year OSRa, RSRb, ASRc, RSRd for Connecticut registry for 1981–1985 and 1991–1995 cohorts with 95% confidence intervals in brackets Connecticut Year 1981–1985 1991–1995 OSRa RSRb ASRc RSRd OSRa RSRb ASRc RSRd 10 6.4 (4.6–8.2) 9.1 (6.7–11.5) 13.0 (12.2–13.8) 16.7 (15.7–17.7) 7.2 (3.7–10.7) 9.7 (6.4–13.0) 15.6 (14.6–16.6) 20.7 (19.5–21.9) 15 3.2 (2.0–4.4) 5.7 (3.5–7.9) 4.4 (4.0–4.8) 6.4 (6.0–6.8) N.A. N.A. 5.8 (5.4–6.2) 8.9 (6.2–9.5) OSRa = overall survival rate calculate by Kaplan-Meier method RSRb = relative survival rate calculated using SEER*Stat 5.0 ASRc = absolute survival rate calculated by period analysis RSRd = relative survival rate calculated by period analysis N.A. = not available Table 3 10- and 15-year OSRa, RSRb, ASRc, RSRd for San Francisco-Oakland registry for 1981–1985 and 1991–1995 cohorts with 95% confidence intervals in brackets San Francisco-Oakland Year 1981–1985 1991–1995 OSRa RSRb ASRc RSRd OSRa RSRb ASRc RSRd 10 4.6 (2.8–6.4) 6.6 (4.2–9.0) 13.3 (12.3–14.3) 16.9 (15.7–18.1) 8.8 (5.7–11.9) 11.4 (7.9–14.9) 14.7 (13.7–15.7) 18.4 (17.2–19.6) 15 2.4 (1.2–3.6) 4.1 (1.9–6.3) 4.6 (4.2–5.0) 6.6 (6.0–7.2) N.A. N.A. 5.3 (4.9–5.7) 7.4 (6.8–8.0) OSRa = overall survival rate calculate by Kaplan-Meier method RSRb = relative survival rate calculated using SEER*Stat 5.0 ASRc = absolute survival rate calculated by period analysis RSRd = relative survival rate calculated by period analysis N.A. = not available Table 4 Cancer sites with survival times demonstrated to follow the lognormal distribution in the literature as at year 2004 Cancer sites Author Head and neck cancer Berg [25], Mould[7] Nasal sinus cancer Berg [25] Mouth and throat cancer Boag [3] Mouth Berg [25] Thyroid Tai*[26] Larynx, tongue Mould & Tai*[27] Non small cell lung cancer Berg [25] Small cell lung cancer Tai*[10] Intraocular melanoma Gamel [8] Cutaneous melanoma Gamel [9] Breast cancer Boag [3], Berg [25], Rutqvist [5,15], Gamel [28,29], Haybittle [30], Royston [31], Tai*[12] Bone sarcomas Berg [25] Cancer of uterine cerivx Mould & Boag*[6], Berg [25] Ovarian cancer Berg [25], Tai*[32], Royston [31] Hypernephroma Berg [25] Bladder cancer Berg [25] Prostate Cancer Mould & Tai*[33] Gastric cancer Berg [25], Maetani [34] Lymphoma Berg [25] Chronic leukemia Tivey [35] Brain tumors Berg [25] 42 SEER cancer sites Tai [36] SEER, Surveillance, Epidemiology, and End Results; *, phase 2 validation also performed, i.e. concordance between Kaplan-Meier method and lognormal model. ==== Refs Boag JW The presentation and analysis of the results of radiotherapy. Part I. Introduction Br J Radiol 1948 21 128 138 Boag JW The presentation and analysis of the results of radiotherapy. Part II. Mathematical theory Br J Radiol 1948 21 189 203 Boag JW Maximum likelihood estimates of the proportion of patients cured by cancer therapy J R Stat Soc (Ser B) 1949 11 15 53 Kaplan EL Meier P Nonparametric estimation from incomplete observations J Am Stat Assoc 1958 53 457 481 Rutqvist LE On the utility of the lognormal model for analysis of breast cancer survival in Sweden 1961–1973 Br J Cancer 1985 52 875 883 4074639 Mould RF Boag JW A test of several parametric statistical models for estimating success rate in the treatment of carcinoma cervix uteri Br J Cancer 1975 32 529 550 1212415 Mould RF Hearnden T Palmer M White GC Distribution of survival times of 12,000 head and neck cancer patients who died with their disease Br J Cancer 1976 34 180 190 962995 Gamel JW Greenberg RA McLean IW A stable linear algorithm for fitting the lognormal model to survival data Comput Biomed Res 1988 21 38 47 3345651 10.1016/0010-4809(88)90040-7 Gamel JW George SL Edwards MJ Seigler HF The long-term clinical course of patients with cutaneous melanoma Cancer 2002 95 1286 1293 12216097 10.1002/cncr.10813 Tai P Tonita JYuE Skarsgard D A 20-year follow-up study of the long-term survival of limited stage small cell lung cancer and an overview of prognostic and treatment factors Int J Radiat Oncol Biol Phys 2003 56 626 633 12788167 10.1016/S0360-3016(03)00070-1 Surveillance, Epidemiology, and End Results Program Public Use Data (1973–2000) National Cancer Institute, released April 2003. Bethesda: National Cancer Institute Tai P Yu E Skarsgard D Tonita J Validation of the lognormal model for prediction of long-term survival rates from short-term follow-up data in stages III and IV breast cancer: a 22-year follow-up study Curr Oncol 2003 10 191 194 Arndt V Talback M Gefeller O Hakulinen T Brenner H Modification of SAS macros for a more efficient analysis of relative survival rates Eur J Cancer 2004 40 778 779 15010081 10.1016/j.ejca.2003.12.013 Hakulinen T Cancer survival corrected for heterogeneity in patient withdrawal Biometrics 1982 38 933 942 7168796 Rutqvist LE Wallgren A Nilsson BO Is breast cancer a curable disease? A study of 14,731 women with breast cancer from the cancer registry of Norway Cancer 1984 53 1793 1800 6697318 Rudan I Skoric T Rudan N Breast cancer prognosis. III. Prognostic factors in patients with distant metastases (M1) at the time of diagnosis Acta Med Croatica 1994 48 171 174 7534519 Chapman JW Fish EB Link MA Competing risks analyses for recurrence from primary breast cancer Br J Cancer 1999 79 1508 1513 10188898 10.1038/sj.bjc.6690240 Engel J Eckel R Kerr J Schmidt M Furstenberger G Richter R Sauer H Senn HJ Holzel D The process of metastasisation for breast cancer Eur J Cancer 2003 39 1794 1806 12888376 10.1016/S0959-8049(03)00422-2 Michaelson JS Silverstein M Wyatt J Weber G Moore R Halpern E Kopans DB Hughes K Predicting the survival of patients with breast carcinoma using tumor size Cancer 2002 95 713 723 12209713 10.1002/cncr.10742 Tubiana M Koscielny S Natural history of human breast cancer: recent data and clinical implications Breast Cancer Res Treat 1991 18 125 140 1756255 Douglas JRS Significance of the size distribution of blood-borne metastases Cancer 1971 27 379 390 5541952 Schulz W Hort W The distribution of metastases in the liver. A quantitative postmortem study Virchows Arch A: Pathol Anat Histol 1981 394 89 96 7336575 Yamanami H Chiba R Kobari M Takahashi T Total number and size distribution of hepatic metastases of carcinoma Anal Quant Cytol Histol 1999 21 216 226 10560494 Kendal WS The size distribution of human hematogenous metastases J Theor Biol 2001 211 29 38 11407889 10.1006/jtbi.2001.2326 Berg JW The distribution of cancer deaths in time. A survey test of the lognormal model Br J Cancer 1965 19 695 711 5862653 Tai P Shiels R Analytic epidemiology: risk and survival prediction for thyroid cancer Curr Oncol 2003 10 195 201 Mould RF Lederman M Tai P Wong JKM Methodology to predict long-term cancer survival from short-term data using Tobacco Cancer Risk and Absolute Cancer Cure Models Phys Med Biol 2002 47 3893 3924 12476973 10.1088/0031-9155/47/22/301 Gamel JW Vogel RL Valagussa P Bonadonna G Parametric survival analysis of adjuvant therapy for stage II breast cancer Cancer 1994 74 2483 2490 7923004 Gamel JW Meyer JS Feuer E Miller BA The impact of stage and histology on the long-term clinical course of 163,808 patients with breast cancer Cancer 1996 77 1459 1464 8608529 10.1002/(SICI)1097-0142(19960415)77:8<1459::AID-CNCR6>3.3.CO;2-7 Haybittle JL The estimation of the proportion of patients cured after treatment for cancer of the breast Br J Radiol 1959 32 725 733 14400306 Royston P The lognormal distribution as a model for survival time in cancer, with an emphasis on prognostic factors Stat Neerlandica 2001 55 89 104 10.1111/1467-9574.00158 Tai P Shiels R Brydon B Liem SK A 32-year follow-up verification of the application of the Lognormal Model to predict long-term survival rates by using short-term follow-up data in ovarian cancer Proceedings of Canadian Association of Radiation Oncologists meeting, 2003 Radioth Oncol 2003 69 S31 (abstr 125) Mould RF Tai THP Wong JKM Bruggmoser G, Mould RF, Tai THP, Mate TP Validation of Lognormal Prediction Models for estimating long-term survival rates from short-term follow-up data for cancer of the prostate patients registered by the USA SEER Program Prostate cancer review 2003 Chapter 19 Munich: W Zuckschwerdt 135 152 Maetani S Tobe T Hirakawa A Kashiwara S Kuramoto S Parametric survival analysis of gastric cancer patients Cancer 1980 46 2709 2716 7448710 Tivey H The prognosis for survival in chronic granulocytic and lymphocytic leukemia Am J Roentgenol 1954 72 68 93 Tai P Tonita J Yu E Skarsgard D Verification of lognormality of survival time for 42 cancer sites in the SEER database and early estimation of their statistical cure rates Proceedings of the 29th Annual Scientific Meeting of Clinical Oncological Society of Australia (COSA) & 2nd Sino-Australian-New Zealand Conference on Surgical Oncology, Sydney, Australia 2002 abstr Pearlman AW Breast cancer: Influence of growth rate on prognosis and treatment evaluation. A study based on mastectomy scar recurrences Cancer 1976 38 1826 1833 991096 von Fournier D Weber E Hoeffken W Bauer M Kubli F Barth V Growth rate of 147 mammary carcinomas Cancer 1980 45 2198 2207 7370960 Debonis D Terz JJ Eldar S Hill LR Survival of patients with metastatic breast cancer diagnosed between 1955 and 1980 J Surg Oncol 1991 48 158 163 1943110 Dickman PW Hakulinen T Luostarinen T Pukkala E Sankila R Söderman B Teppo L Survival of cancer patients in Finland 1955–1994 Acta Oncol 1999 38 1 103 10225326 Todd M Shoag M Cadman E Survival of women with metastatic breast cancer at Yale from 1920 to 1980 J Clin Oncol 1983 1 406 408 6668509 Farrow DC Samet JM Hunt WC Regional variation in survival following the diagnosis of cancer J Clin Epidemiol 1996 49 843 847 8699202 10.1016/0895-4356(96)00176-X Maggard MA Thompson JE Ko CY Why do breast cancer mortality rates vary across states? Am Surg 2003 69 59 62 12575783 Goodwin JS Freeman JL Mahnken JD Freeman DH Nattinger AB Geographic variations in breast cancer survival among older women: implications for quality of breast cancer care J Gerontol A Biol Sci Med Sci 2002 57 M401 406 12023271 Goodwin JS Freeman JL Freeman D Nattinger AB Geographic variations in breast cancer mortality: do higher rates imply elevated incidence or poorer survival? Am J Public Health 1998 88 458 460 9518983

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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-241531571410.1186/1471-244X-4-24Research ArticleProspective open-label study of add-on and monotherapy topiramate in civilians with chronic nonhallucinatory posttraumatic stress disorder Berlant Jeffrey L [email protected] Private practice, Boise, ID, USA2004 18 8 2004 4 24 24 7 6 2004 18 8 2004 Copyright © 2004 Berlant; licensee BioMed Central Ltd.2004Berlant; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In order to confirm therapeutic effects of topiramate on posttraumatic stress disorder (PTSD) observed in a prior study, a new prospective, open-label study was conducted to examine acute responses in chronic, nonhallucinatory PTSD. Methods Thirty-three consecutive newly recruited civilian adult outpatients (mean age 46 years, 85% female) with DSM-IV-diagnosed chronic PTSD, excluding those with concurrent auditory or visual hallucinations, received topiramate either as monotherapy (n = 5) or augmentation (n = 28). The primary measure was a change in the PTSD Checklist-Civilian Version (PCL-C) score from baseline to 4 weeks, with response defined as a ≥ 30% reduction of PTSD symptoms. Results For those taking the PCL-C at both baseline and week 4 (n = 30), total symptoms declined by 49% at week 4 (paired t-test, P < 0.001) with similar subscale reductions for reexperiencing, avoidance/numbing, and hyperarousal symptoms. The response rate at week 4 was 77%. Age, sex, bipolar comorbidity, age at onset of PTSD, duration of symptoms, severity of baseline PCL-C score, and monotherapy versus add-on medication administration did not predict reduction in PTSD symptoms. Median time to full response was 9 days and median dosage was 50 mg/day. Conclusions Promising open-label findings in a new sample converge with findings of a previous study. The use of topiramate for treatment of chronic PTSD, at least in civilians, warrants controlled clinical trials. ==== Body Background Posttraumatic stress disorder (PTSD) is a difficult-to-treat condition that over a lifetime affects approximately 10% of the general population [1]. The condition develops after traumatic events such as combat, terror activities, disaster, or rape, and has 3 main features: (1) reexperiencing the trauma through recollection, dreams, and reliving, (2) avoidance of thoughts, activities, and emotions associated with the trauma, and (3) hyperarousal [2]. PTSD is usually a chronic disorder, with one third of patients displaying symptoms for ≥ 10 years after experiencing the traumatic event [3,4]. Generally the response to pharmacotherapy has been poor, with many patients completely unresponsive and others only marginally responsive [5]. Some tricyclic antidepressants, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors have demonstrated efficacy in double-blind trials [3]. The complex neurobiology of PTSD involves a number of systems, including dopaminergic, serotonergic, sympathetic, hypothalamic-pituitary-adrenal, and various anatomic regions of the amygdala and other parts of the limbic system [3]. It has been suggested that after traumatic events limbic nuclei may become kindled or abnormally sensitized [6], leading to increased susceptibility for psychic and physical arousal and psychiatric disturbance [2]. Because of the suggested involvement of the kindling phenomenon, several anticonvulsants have been assessed in the treatment of PTSD, including carbamazepine, valproate, lamotrigine, gabapentin, tiagabine, and topiramate [3]. Topiramate has a broad spectrum of pharmacologic properties, including Na+ channel blockade [7-11], inhibition of some high voltage-activated Ca2+ channels [12], enhanced γ-aminobutyric acid (GABA) neuroinhibition at novel GABAA receptors [13,14], glutamate inhibition at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors [15,16], and promotion of protein phosphorylation of neuronal conductance channels [15]. These properties, together with inhibitory activity in animal kindling models [17,18], suggest that topiramate may have therapeutic potential in PTSD. Treatment with topiramate has been reported to improve reexperiencing symptoms associated with civilian PTSD [6]. Results from that study, which included a number of patients who were classified as treatment resistant, suggested that topiramate fully or partially suppressed both intrusions (distressing recollections or nonhallucinatory flashbacks) and nightmares, if present, in 89% of patients with nonhallucinatory PTSD. Although the DSM-IV definition of PTSD lists hallucinations among reexperiencing symptoms [19], because a few patients displayed varying degrees of impaired reality testing of hallucinations, it was difficult to determine if hallucinations were due to PTSD, to an independent psychotic disorder, or, in some individuals, to both. Although psychotic variants of PTSD have been reported in as many as 40% of veterans with chronic combat-associated PTSD without evidence of a primary psychotic disorder [20,21], it is also possible for patients with psychotic disorders to have comorbid PTSD, leaving questions about whether specific hallucinations should be attributable to the PTSD reexperiencing cluster or to a psychotic disorder. Because the prior study found a more robust effect in the group of PTSD patients without hallucinations [6], it was decided to focus further on the core group of PTSD patients without ambiguous symptoms confounded by the possible presence of independent psychotic disorders. Based on these earlier results of topiramate in PTSD, a fully prospective open-label study was begun to see whether the previously observed signal of a therapeutic benefit of topiramate for chronic civilian PTSD could be confirmed. None of the patients reported in the earlier study were included in the present study. In order to address methodological limitations of the prior study, the current study is modified in 4 major ways: (1) it excludes patients with hallucinations, (2) it limits the study to 12 weeks, (3) it prospectively employs the PTSD Checklist-Civilian Version (PCL-C) as a validated clinical assessment instrument in all patients, and (4) it determines a clinical response rate as of week 4, using a conventional, predetermined definition for positive response as a ≥ 30% improvement in a standardized clinical rating scale. Methods This sample included all consecutive adults (n = 33) meeting the DSM-IV criteria for chronic civilian PTSD in an outpatient private practice who started open-label topiramate between January 2000 and November 2002 in the course of clinical care. The study excluded patients with concurrent auditory or visual hallucinations associated with either PTSD or a possible psychotic disorder. Topiramate, administered either as monotherapy (n = 5) or added to the patients' existing therapeutic regimen (n = 28) (Table 1), was initiated at a starting dosage of 12.5 to 25 mg/day and increased in 25- to 50-mg/day increments every 3 to 4 days as tolerated until a clinical response was achieved. Table 1 Patient characteristics All subjects N = 33 Mean age, years ± SD 46 ± 6.5  Range 29–55 Sex, %  Women 85  Men 15 Mean age at onset of PTSD, years ± SD 29 ± 15  Range 2–53 Mean duration of PTSD history, years ± SD 18 ± 15  Range 0–46 Patients with comorbid disorders, N (%)  Bipolar disorder 10 (30)  Major depressive disorder 21 (64)  Substance abuse   Current 3 (9)   In past 5 (15) SD =standard deviation; PTSD = posttraumatic stress disorder. The study was performed according to the Declaration of Helsinki. Each participant provided verbal informed consent based on disclosure of off-label usage, the availability of alternative medications for PTSD such as sertraline and paroxetine, and expected adverse events and risks. Verbal consent was deemed appropriate and sufficient because of the nonexperimental nature of the treatment. Because this was a descriptive study using aggregate data gathered in the course of treating patients in clinical practice for their own benefit and not an experimental design, there was no protocol that required Institutional Review Board approval. Patients were asked to measure their symptoms using standardized clinical instruments already routinely used as a part of individual care. The study included no features of clinical studies, such as randomization, concealment of treatment, or surrender of patient decision-making rights, as an integral part of conducting the study. The primary outcome was a change in PTSD symptoms calculated from change in PCL-C score from baseline through 4 weeks of treatment [22]. The PCL-C, a validated self-report instrument, strongly correlates (k~0.90) with the Clinician-Administered PTSD Scale (CAPS), a structured clinical interview widely used in PTSD studies [23]. The PCL-C is scored on a Likert scale of 1 to 5 (1 = not at all; 5 = extremely) for each of 17 items that cover all 3 symptom clusters (B, reexperiencing; C, avoidance/numbing; D, hyperarousal), yielding a score range of 17 to 85. A total score of ≥ 50 meets the threshold for active PTSD. In accordance with conventional usage, a 30% reduction in clinical symptoms of PTSD was defined as a positive clinical response. The secondary measures included were Cluster B symptoms of reexperiencing of trauma, including trauma-related intrusions and nightmares. Secondary measures were limited to Cluster B symptoms because of the practical difficulty patients in clinical practice can have linking a symptom (such as avoidance, numbing, hyperarousal, insomnia, or startling) specifically to a trauma as opposed to some other source, as required by the DSM-IV definitions. Also, Cluster B symptoms appeared to be among the most distressing and salient for many patients, thereby making tracking these distinctive symptoms comparatively easy. Data on patient demographics, concomitant medication, comorbidities, primary trauma, and duration of PTSD were collected at baseline. Assessments were made through clinical interviews over a 12-week period. Cessation of reexperiencing symptoms was considered partial if subjects reported a definite reduction in intensity and frequency of nightmares or intrusive recollections or flashbacks. Full cessation was defined as complete elimination of nightmares and intrusions for a sustained period. Adverse effects were recorded if they resulted in treatment discontinuation. Statistical analyses were performed using Jandel SigmaStat® Version 2.0 (Jandel Corporation, San Rafael, CA, USA). A single paired t-test of before and after total PCL-C scores was used to analyze the data. Subscale scores for PCL-C Clusters B, C, and D were also evaluated. Results Patient characteristics Baseline patient characteristics and concomitantly administered medications are listed in Tables 1 and 2, respectively. All monotherapy patients were in the nonbipolar subgroup. Primary traumas for which symptoms were reported are detailed in Table 3. Of these, 9 were of childhood onset, with 4 involving childhood physical assault, 3 unwanted sex or sexual assault, and 2 sudden unexpected or violent deaths. Table 2 Concomitant medications Medication type Number of patients (N = 33) SSRIs 12 Benzodiazepines 10 Stimulants 6 Atypical neuroleptics 3 Gabapentin 3 Lamotrigine 5 Mirtazapine 3 Venlafaxine 3 Verapamil 1 Other 8 Monotherapy 5 SSRIs = selective serotonin reuptake inhibitors. Table 3 Primary traumas Trauma type Number of patients, N = 33 Unwanted sex 11 Physical assault 6 Sudden violent death 4 Sudden unexpected death of loved one 3 Sexual assault 2 Fire/explosion 1 Weapon assault 1 Combat 1 Life-threatening illness 1 Other 3 Primary measures Reduction in PCL-C symptoms Ninety-one percent of patients (30/33) with baseline PCL-C measurements completed a PCL-C at week 4. In those patients, the total score was 62.6 at baseline and 40.3 at week 4. In addition, the scores for reexperiencing, avoidance, and hyperarousal clusters were 18.2, 25.1, and 19.2 at baseline and 11.2, 17.2, and 12.3 at week 4, respectively. To calculate percentage reduction in PTSD symptoms, to correct for an absence of symptoms being scored as "1" for each item, the change in score between baseline and week 4 was divided by the baseline score minus the minimum score for no symptoms. Therefore, the change in total symptoms was calculated as (baseline – week 4)/(baseline – 17). Total symptoms declined significantly (49%) by week 4 (paired t-test, P < 0.001). In addition, subscale symptoms for reexperiencing (Cluster B), avoidance/numbing (Cluster C), and hyperarousal (Cluster D) decreased by 53%, 43%, and 48%, respectively, from baseline (Figure 1). By the end of week 4, 70% of all entrants in the study (n = 33) and 77% of those with both baseline and week 4 PCL-C scores (n = 30) were positive responders, defined as a ≥ 30% improvement in symptoms. Figure 1 Mean percentage symptom reduction at week 4. Symptom reduction in those 30 subjects who completed a PCL-C at baseline and after 4 weeks. *Paired t-test, P < 0.001 versus baseline. PTSD = posttraumatic stress disorder; PCL-C = PTSD Checklist-Civilian Version. Predictors of reduction in PCL-C symptom improvement Comparisons for bipolar versus nonbipolar patients (50% versus 49%, analysis of variance [ANOVA]), duration of symptoms (r = 0.37), age at onset of PTSD (r = -0.36), severity of baseline PCL-C score (r = 0.02), age (r = -0.02), female sex (49% versus 50%, ANOVA), and monotherapy versus add-on medication administration (53% versus 48%, ANOVA) found none significantly associated with reduction in PCL-C symptoms. Secondary measures Results for combined suppression of nightmares and intrusions are shown in Table 4 Table 4 Secondary efficacy measures Combined suppression of nightmares and intrusions Responder status, n (%)  Full 26/33 (79%)  Partial 3/33 (9%)  None 4/33 (12%) Mean time to response, days ± SD, (range)  Full response (n = 25) 15 ± 18 (1–83)  Partial response (n = 17) 11 ± 13 (2–46) Median time to full response, days 9 Median time to partial response, days 5 Mean dosage at time of response, mg/day  Full response 60 ± 47  Partial response 32 ± 15 Median dosage at time of response, mg/day  Full response 50  Partial response 25 Modal daily dosage for full response, mg/day 25  % of patients in the 12.5–50-mg/day range 65 Modal dosage at time of partial response, mg/day 25  % of patients in the 12.5–50-mg/day range 100 Improvements in nightmares Full cessation of nightmares 17/18 (94%) Improvements in intrusions Full cessation of intrusions 26/33 (79%) Partial improvement 3/33 (9%) No improvement 4/33 (12%) SD = standard deviation. Of the 33 patients who entered the study, 79% fully ceased having reexperiencing symptoms with a mean time to full cessation of 15 days. The onset of full cessation for all patients occurred at ≤ 200 mg/day and the onset of partial cessation for all patients at ≤ 50 mg/day. Ninety-four percent of patients with nightmares and 79% of patients with intrusions at baseline reported full cessation at the end of week 4. Discontinuations Serious adverse events There were no serious adverse events during this study. Of the 33 patients who entered the study, 7 (21%) discontinued due to adverse events and 5 (15%) for other reasons. The single most common adverse event resulting in discontinuation was intolerable nervous system overstimulation (panic/nervousness/overstimulation/ shakiness), reported by 3 patients. Other adverse events resulting in discontinuation included clumsiness (n = 2), cognitive impairment (n = 1), and severe headache (n = 1). Additional reasons for discontinuation included personal choice (n = 1) and lack of relapse of PTSD symptoms after interrupting medication (n = 4). Of the 12 patients that discontinued, 54% (n = 7) fully ceased having reexperiencing symptoms of nightmares/intrusions and 15% (n = 2) partially ceased having reexperiencing symptoms of nightmares/intrusions. The median time to termination was 25.5 days (range, 4–77). Predictors of discontinuation There were no statistically significant predictors of panic-like adverse events resulting in discontinuation from the study (age, sex, symptom duration, age at onset, bipolarity, substance abuse, baseline severity of illness), although the coadministration of a benzodiazepine nearly reached significance (Fisher exact test, P = 0.05). No monotherapy patients discontinued because of overstimulation/panic-like responses, so the possibility of drug interactions or conditions such as comorbid panic disorder contributing to this reaction should be considered in future investigations. Discussion Results of this prospective open-label study suggest that the use of topiramate for the treatment of chronic PTSD in civilians is indeed promising and warrants controlled clinical trials. Topiramate was able to improve symptoms of reexperiencing, avoidance, and hyperarousal by 49% overall, compared with baseline, and decreased intrusions in 94% of patients and nightmares in 79% of those with this symptom. Although there are numerous other reports describing the use of antiepileptic agents for the treatment of PTSD, most are case reports and case series, and some lack the use of standardized PTSD scales. The first published trials using anticonvulsants date back almost 20 years, when a 5-week trial of carbamazepine in 10 combat veterans produced a 36% reduction in a nonvalidated interviewer-scored instrument [24]. Studies on adjunctive therapy with valproate reported improved global scores in avoidance and hyperarousal, but not reexperiencing symptoms in veterans over a 14-month period [25], whereas a subsequent 8-week open-label trial reported a small, 17% reduction in CAPS score in 13 subjects with significant reductions in all 3 cluster criteria [26]. Two studies in which valproate was used as monotherapy reported varying results, with one showing a 30% reduction in total CAPS scores in 28 veterans [27], whereas the other in 10 civilian patients with non-combat-related PTSD showed no improvement [28].The only double-blind placebo-controlled trial evaluating an anticonvulsant in PTSD was conducted with lamotrigine [29]. Overall, after 10 weeks of treatment, 5 of 10 subjects were characterized as responders, compared with 1 of 4 subjects receiving placebo. However, a last observation carried forward analysis revealed only a 23% decrease in mean pretreatment scores with lamotrigine, compared with 20% in the placebo group. A retrospective clinical series of adjunctive gabapentin therapy in 30 consecutive patients with PTSD assessed improvement of target symptoms such as nightmares, insomnia, and irritability. It was reported that the majority of patients (77%) showed moderate or greater improvement in duration of sleep, and most notably a decrease in the frequency of nightmares [30]. These studies highlight the need for double-blind placebo-controlled trials using standardized and validated questionnaires. It is encouraging that the results of this current study with topiramate were consistent with the earlier, preliminary data obtained in patients with PTSD. Percentage responders (77% versus 75%), median time to full response (9 d versus 8 d), suppression of intrusions and nightmares (88% versus 89%), and discontinuation rate (39% versus 32%) were all similar. Overall, the open-label experience with topiramate, based on patient statements and self-rating reports, suggests that the drug may have a rapid rate of response with limited risk of clinically significant adverse events and no evidence of tolerance developing over time [6,31]. Moreover, from topiramate studies in epilepsy [Topamax prescribing information] it is known that the drug is not associated with the cardiac, pancreatic, and hematologic toxicity found with valproate or carbamazepine [3]. In addition, topiramate is not associated with weight gain, a side effect reported with valproate that can predispose patients to diabetes mellitus [32]. Topiramate also offers potential clinical advantages over serotonin reuptake inhibitors (SSRIs) because it does not appear to destabilize mood in patients with comorbid bipolar disorder and is relatively devoid of common adverse effects of SSRIs, such as sexual dysfunction, weight gain with chronic use, or sedation when used as monotherapy. Limitations of the present study include: (1) the absence of structured assessment data following week 4 to test the maintenance of response over time, (2) use of self-report instruments that, although validated and correlated with results of structured clinical interviews, may be less accurate than structured interviews, and (3) the absence of clinical assessment scores at the time patients discontinued the trial. From a broader perspective, the greatest limitation of this study is characteristic of all open trials: the lack of standard features of clinical trials such as placebo controls, randomization, and blinding of raters. Conclusions Fundamentally, the findings of current and prior topiramate studies are convergent and consistently signal a potentially beneficial therapeutic effect for all 3 criteria clusters of chronic PTSD in civilian adults. In both studies, there is a positive response in a large proportion of patients, at dosages considerably below the usual anticonvulsant dosage levels of 200 to 600 mg/day, and a rapid onset. The effect appears independent of comorbidity with bipolar disorder, age, sex, duration of symptoms, baseline severity of illness, or administration alone or with other psychotropic medications. The purpose of this study was to prospectively test the results of an earlier open-label trial in a new sample; however, the limitations of those open studies will have to be addressed in double-blind, placebo-controlled studies, which are currently in progress. Competing interests The author is a consultant to and a participant in the Speaker's Bureau for Ortho-McNeil Pharmaceutical, Inc. He also holds a licensing agreement with Ortho-McNeil based on issuance of a United States Government patent for topiramate therapy of PTSD. Author's contributions JLB conceived of the study and its design, coordination, statistical analysis, and drafted the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Breslau N The epidemiology of posttraumatic stress disorder: what is the extent of the problem? J Clin Psychiatry 2001 62 16 22 11495091 van der Kolk BA The psychobiology of posttraumatic stress disorder J Clin Psychiatry 1997 58 16 24 9329447 Iancu I Rosen Y Moshe K Antiepileptic drugs in posttraumatic stress disorder Clin Neuropharmacol 2002 25 225 229 12151910 10.1097/00002826-200207000-00006 Berlant J Antiepileptic treatment of posttraumatic stress disorder Primary Psychiatry 2003 10 41 49 Davidson JR Biological therapies for posttraumatic stress disorder: an overview J Clin Psychiatry 1997 58 29 32 9329449 Berlant J van Kammen DP Open-label topiramate as primary or adjunctive therapy in chronic civilian posttraumatic stress disorder: a preliminary report J Clin Psychiatry 2002 63 15 20 11838620 DeLorenzo RJ Sombati S Coulter DA Effects of topiramate on sustained repetitive firing and spontaneous recurrent seizure discharges in cultured hippocampal neurons Epilepsia 2000 41 S40 44 10768299 Zona C Ciotti MT Avoli M Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells Neurosci Lett 1997 231 123 126 9300637 10.1016/S0304-3940(97)00543-0 McLean MJ Bukhari AA Wamil AW Effects of topiramate on sodium-dependent action-potential firing by mouse spinal cord neurons in cell culture Epilepsia 2000 41 S21 24 10768295 Taverna S Sancini G Mantegazza M Franceschetti S Avanzini G Inhibition of transient and persistent Na+ current fractions by the new anticonvulsant topiramate J Pharmacol Exp Ther 1999 288 960 968 10027832 Wu SP Tsai JJ Gean PW Frequency-dependent inhibition of neuronal activity by topiramate in rat hippocampal slices Br J Pharmacol 1998 125 826 832 9831921 Zhang X Velumian AA Jones OT Carlen PL Modulation of high-voltage-activated calcium channels in dentate granule cells by topiramate Epilepsia 2000 41 S52 60 10768302 White HS Brown SD Woodhead JH Skeen GA Wolf HH Topiramate enhances GABA-mediated chloride flux and GABA-evoked chloride currents in murine brain neurons and increases seizure threshold Epilepsy Res 1997 28 167 179 9332882 10.1016/S0920-1211(97)00045-4 White HS Brown SD Woodhead JH Skeen GA Wolf HH Topiramate modulates GABA-evoked currents in murine cortical neurons by a nonbenzodiazepine mechanism Epilepsia 2000 41 S17 20 10768294 Gibbs JW IIISombati S DeLorenzo RJ Coulter DA Cellular actions of topiramate: blockade of kainate-evoked inward currents in cultured hippocampal neurons Epilepsia 2000 41 S10 S16 10768293 Skradski S White HS Topiramate blocks kainate-evoked cobalt influx into cultured neurons Epilepsia 2000 41 S45 S47 10768300 Wauquier A Zhou S Topiramate: a potent anticonvulsant in the amygdala-kindled rat Epilepsy Res 1996 24 73 77 8796355 10.1016/0920-1211(95)00105-0 Amano K Hamada K Yagi K Seino M Antiepileptic effects of topiramate on amygdaloid kindling in rats Epilepsy Res 1998 31 123 128 9714503 10.1016/S0920-1211(98)00021-7 DSM-IV: Diagnostic and Statistical Manual of Mental Disorders 1994 4 Washington, DC: American Psychiatric Association Hamner MB Psychotic features and combat-associated PTSD Depress Anxiety 1997 5 34 38 9250439 10.1002/(SICI)1520-6394(1997)5:1<34::AID-DA6>3.0.CO;2-5 Hamner MB Frueh BC Ulmer HG Arana GW Psychotic features and illness severity in combat veterans with chronic posttraumatic stress disorder Biol Psychiatry 1999 45 846 852 10202572 10.1016/S0006-3223(98)00301-1 Blanchard EB Jones-Alexander J Buckley TC Forneris CA Psychometric properties of the PTSD Checklist (PCL) Behav Res Ther 1996 34 669 673 8870294 10.1016/0005-7967(96)00033-2 Weathers FW Keane TM Davidson JR Clinician-administered PTSD scale: a review of the first ten years of research Depress Anxiety 2001 13 132 156 11387733 10.1002/da.1029 Lipper S Davidson JR Grady TA Edinger JD Hammett EB Mahorney SL Cavenar JO Jr Preliminary study of carbamazepine in post-traumatic stress disorder Psychosomatics 1986 27 849 854 3543990 Fesler FA Valproate in combat-related posttraumatic stress disorder J Clin Psychiatry 1991 52 361 364 1894587 Clark RD Canive JM Calais LA Qualls CR Tuason VB Divalproex in posttraumatic stress disorder: an open-label clinical trial J Trauma Stress 1999 12 395 401 10378177 10.1023/A:1024797014210 Petty F Davis LL Nugent AL Kramer GL Teten A Schmitt A Stone RC Valproate therapy for chronic, combat-induced posttraumatic stress disorder J Clin Psychopharmacol 2002 22 100 101 11799355 10.1097/00004714-200202000-00021 Otte C Wiedemann K Yassouridis A Kellner M Valproate monotherapy in the treatment of civilian patients with non-combat-related posttraumatic stress disorder: an open-label study J Clin Psychopharmacol 2004 24 106 108 14709964 10.1097/01.jcp.0000106234.36344.a4 Hertzberg MA Butterfield MI Feldman ME Beckham JC Sutherland SM Connor KM Davidson JR A preliminary study of lamotrigine for the treatment of posttraumatic stress disorder Biol Psychiatry 1999 45 1226 1229 10331117 10.1016/S0006-3223(99)00011-6 Hamner MB Brodrick PS Labbate LA Gabapentin in PTSD: a retrospective, clinical series of adjunctive therapy Ann Clin Psychiatry 2001 13 141 146 11791951 10.1023/A:1012281424057 Berlant JL Topiramate in posttraumatic stress disorder: preliminary clinical observations J Clin Psychiatry 2001 62 60 63 11495099 Corman CL Leung NM Guberman AH Weight gain in epileptic patients during treatment with valproic acid: a retrospective study Can J Neurol Sci 1997 24 240 244 9276111

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BMC Psychiatry. 2004 Aug 18; 4:24

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==== Front BMC PsychiatryBMC Psychiatry1471-244XBioMed Central London 1471-244X-4-251531765310.1186/1471-244X-4-25Research ArticleThe link between thyroid autoimmunity (antithyroid peroxidase autoantibodies) with anxiety and mood disorders in the community: a field of interest for public health in the future Carta Mauro Giovanni [email protected] Andrea [email protected] Maria Carolina [email protected] Sergio [email protected] Mariangela [email protected] Claudia [email protected] Bernardo [email protected]'Osso Liliana [email protected] Stefano [email protected] Department of Public Health, Division of Psychiatry, University of Cagliari, Italy2 Department of Internal Medicine, University of Cagliari, Italy3 Department of Psychiatry, Neurobiology, Pharmacology, Biotechnology, University of Pisa, Italy4 Department of Public Health, University of Cagliari, Italy2004 18 8 2004 4 25 25 5 4 2004 18 8 2004 Copyright © 2004 Carta et al; licensee BioMed Central Ltd.2004Carta et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background To evaluate the association between mood and anxiety disorders and thyroid autoimmunity in a community sample. Methods: A community based sample of 222 subjects was examined. Psychiatric diagnoses were formulated using the International Composite Diagnostic Interview Simplified (CIDIS), according to DSM-IV criteria. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). Results 16.6% of the overall sample had an anti-TPO value above the normal cut-off. Subjects with at least one diagnosis of anxiety disorders (OR = 4.2, C.L. 95% 1.9–38.8) or mood disorders (OR = 2.9, Cl 95% 1.4–6.6, P < 0.011) were positive for serum anti-TPO more frequently than subjects without mood or anxiety disorders. A statistically significant association with anti-TPO+ was found in Anxiety Disorder Not Otherwise Specified (OR = 4.0, CL 95% 1.1–15.5), in Major Depressive Episode (OR = 2.7, CL 95% 1.1–6.7) and Depressive Disorder Not Otherwise Specified (OR = 4.4, S CL 95% 1–19.3). Conclusions The study seems to suggest that individuals in the community with thyroid autoimmunity may be at high risk for mood and anxiety disorders. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. Should our results be confirmed, the findings may be of great interest for future preventive and case finding projects. ==== Body Background Autoimmune thyroid disease may be linked to depression [1] and anxiety [2]. Autoimmune disease and depression are not uncommon: the prevalence of autoimmune thyroid disease in the community ranged from 4 to 25% [3] and lifetime prevalence of Major Depressive Disorder ranged from 6 to 17% [4]. Thus the association may have a great relevance in terms of public health and prevention. The purpose of this investigation was to evaluate the relationship between mood and anxiety disorders and thyroid autoimmunity in a community survey. This research was carried out on the data base of two epidemiological studies aimed at defining the prevalence of psychiatric [5] and thyroid diseases [6] in Sardinia. On planning these surveys, researchers agreed to evaluate a representative sub-sample of a defined geographical area common to both endocrinological and psychiatric epidemiological surveys. This paper present the results of the cross psychiatric and endocrinological evaluation from the common areas of the two surveys. Methods The sample was extracted by randomization (1/10) subsequent to stratification according to age and sex, from the records of 2 Sardinian villages. Probands were interviewed face to face in their homes by specifically trained physicians. Two standardized forms were used to acquire information concerning: demographic data, state of health and use of social and health services. Psychiatric diagnosis was made using the Italian Simplified version of the Composite International Diagnostic Interview (CIDIS) [7]. The computer elaboration of data obtained enabled prevalence of psychiatric disorders according to DSM-IV [8] diagnostic criteria to be calculated. Anti-thyroid peroxidase autoantibodies (anti-TPO), considered as the most sensitive and specific marker of thyroid autoimmunity [9] was determined by RIA (Sorin Biomedica Diagnostics, Saluggia, Italy) with a cut-off value of 20 IU/ml. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). FT4 and FT3 were measured by means of a chromatographic method based on separation of free T4 on Lisophase columns (Technogenetics, Milan, Italy; normal values: FT4 6.6–16 pg/ml; FT3 2.8–5.6 pg/ml). TSH was measured by a chemiluminescent method (Ortho-Clinical Diagnostics Amersham, U.K.) with normal values ranging from 0.3–3.0 μU/ml. Thyroid echography was performed using a "real time" echograph (ALOKA Mod SSD 500 with a small parts 7.5). The association of anti- TPO+ with the main diagnoses deriving from CIDIS interview was calculated using Odds Ratio. Statistical significance was calculated using the X2 test in 2 × 2 tables. Odds Ratio confidence intervals were calculated through application of the method of Miettinen [10]. Multivariate Logistic Regression was performed in order to evaluate the possible influences of gender and age on the association between anti-TPO+ and mood or anxiety disorders. The analysis was carried out considering mood (or anxiety) disorders as dependent variable, and anti-TPO+ (presence vs absence), gender (female vs male) and age (≤ 44 vs > 44) and their second order interactions as independent variables, by means of backward stepwise procedure; interactions lacking evidence of association (p > 0.20) were eliminated from the models. Results From a total of 261 subjects identified (age >18 years), 222 (85.1%), 127 females (57.2%), and 95 males (42.7%); over 44 years 127 (57.2%), 79 females (62.2%,) 48 males (37.7%), agreed to take part in the study whilst 20 (8,7%) refused to participate and 19 (7.3%) could not be traced. The final sample did not differ respect to the population of origin with reference to the variables applied in stratification. The lifetime prevalence of anxiety disorders in the sample was: Generalized Anxiety Disorder (GAD) 11.3%, Panic Disorder (PD) 2.7%, Anxiety Disorder Not Otherwise Specified (ADNOS) 5.4%, Social Phobia (SP) 5.4%; 18.5% had been diagnosed with at least one of these anxiety disorders. With regard to mood disorders, Major Depressive Episode (MDE) was present in 14.4%, Dysthymic Disorder (DD) in 2.7%, Depressive Disorder Not Otherwise Specified (DDNOS) in 4.0%; 18.9% had at least one of the above mentioned depressive disorders. 1.1% of the overall sample was affected by hypothyroidism, 16.6% had an anti-TPO value above the normal cut-off (anti-TPO+). Table 1 shows the association between anti-TPO+ and lifetime psychiatric diagnosis. Subjects with at least one lifetime diagnosis of anxiety disorders or one lifetime diagnosis of mood disorders presented anti-TPO+ more frequently than subjects without mood or anxiety disorders. An association with anti-TPO+ was found in ADNOS, in MDE and DDNOS. The attributable risk for ADNOS was 0.54, 0.37 for MDE and 0.34 for DDNOS, other conditions as PD and DD presented very high attributable risk. Table 1 Association between positivity anti-TPO (anti-TPO+), mood and anxiety diagnosis. Diagnosis Prev.nce N (%) Anti-TPO+ N (%) OR IC 95% χ2 P Att.able Risk One Anxiety Diagnosis GAD+PD+SP+ADNOS 41 (18.5) 15 (36.6) 4.2 1.9/38.8 12.6 0.001 0.54 One Mood Diagnosis MDE+DD+DDNOS 42 (18.9) 13 (30.9) 2.9 1.4/6.6 6.4 0.011 0.37 GAD 25 (11.3) 8 (32) 2.7 0.97/7.5 3.6 0.058 0.35 PD 6 (2.7) 3 (50) 5.4 0.7/37.3 2.8 0.096 0.68 SP 12 (5.4) 4 (40) 3.6 0.7/7.6 2.5 0.111 0.52 ADNOS 12 (5.4) 5 (41.7) 4.0 1.1/15.5 3.9 0.045 0.55 MDE 32 (14.4) 10 (31.2) 2.7 1.1/6.7 4.6 0.033 0.34 DD 6 (2.7) 2 (50) 5.2 0.3/16.8 1.3 0.250 0.67 DDNOS 9 (4.0) 4 (44.4) 4.4 1/19.3 3.8 0.049 0.60 GAD: Generalized Anxiety Disorder; PD: Panic Disorder; SP: Social Phobia; ADNOS: Anxiety Disorder Not Otherwise Specified; MDE: Major Depressive Episode; DD: Dysthymic Disorder; DDNOS: Depressive Disorder Not Otherwise Specified. The Multivariate Logistic Regression showed that gender and age do not interact with anti-TPO either in mood (interaction anti-TPO-gender p = 0.97, OR 1.03, CI 95%, 0.19–5.48; interaction anti-TPO-age p = 0.62, OR = 1.52, CI 95%, 0.30–7.77), or in anxiety disorders (interaction anti-TPO-gender p = 0.93, OR = 0.93, CI 95%, 0.18–4.80; interaction anti-TPO-age p = 0.67, OR = 0.70, CI 95%, 0.14–3.64). Interactions were therefore eliminated from the models. The final Logistic Regression models clearly indicated that gender and age do not influence the risk of one mood or anxiety diagnosis either as independent variables or as confounders (Table 2, Table 3 and Table 4). Table 2 Frequency of anti-TPO+ in to the sample according sex and age and one mood diagnosis (OMD) and one anxiety diagnosis (OAD) diagnosis for multivariate logistic regression. Age Sex N anti-TPO+ (%) OMD (%) OMD (%) #anti-TPO+ OAD (%) OAD (%) #anti-TPO+ <45 Female 79 17 (21.5) 17 (21.5) 6 (35.2) 13 (16.4) 6 (35.2) <45 Male 48 8 (16.6) 7 (14.5) 2 (24) 7 (14.5) 3 (37.5) >44 Female 48 7 (14.5) 10 (20.8) 3 (42.8) 14 (29.1) 4 (57.1) >44 Male 47 5 (10.6) 8 (17.2) 2 (40) 7 (15.1) 2 (40) #TPO+ in the sub-group is the total Table 3 Multivariate logistic regression: effect of anti-TPO+ on risk of one mood diagnosis considering gender (female vs male) and age (≤ 44 vs > 44) effects. p OR CI 95% anti-TPO+ vs anti-TPO- 0.01 2.89 1.31–6.38 Gender (F vs M) 0.37 1.38 0.68–2.82 Age (≤ 44 vs > 44) 0.71 1.14 0.57–2.30 Table 4 Multivariate Logistic Regression: effect of anti-TPO+ on risk of one anxiety diagnosis considering gender (Female vs Male) and age (≤ 44 vs > 44) effects. p OR CI 95% anti-TPO+ vs anti-TPO- 0.001 4.50 2.02 – 10.04 Gender (F vs M) 0.23 1.58 0.75 – 3.31 Age (≤ 44 vs > 44) 0.08 1.91 0.92 – 3.96 Discussion The present study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+ in a general population sample which had not been selected from medical or psychiatric health facilities. This association is independent by gender and age. Regarding specific diagnosis, MDE, DDNOS and ADNOS were associated with anti-TPO+. This finding is consistent with several previous clinical studies providing evidence for a significant association of mood disorders or post-partum depression and symptomless autoimmune thyroiditis with or without sub-clinical hypothyroidism [11]. Association between hypothyroidism and mood disorders is however controversial as other authors maintain that bipolar disorders rather than unipolar depression are characterized by an increased risk for the presence of anti-thyroid antibodies [11]. However, a study performed by Fountoulakis and collaborators [1] recently found a link between autoimmune thyroid disease and Unipolar Depression. In this study, compared to control patients all depressive subtypes had significantly higher thyroid binding inhibitory immunoglobulins, and atypical patients had significantly higher thyroid microsomal antibodies. Thyroid function markers Free Triiodothyronine (FT3), Free Thyroxine (FT4), and Thyroid Stimulating Hormone (TSH) were normal in all subjects suggesting that Unipolar Depression might be characterized by a "low-thyroid function syndrome". A sub-clinical dysfunction of axis Thyrotropin Releasing Hormone (TRH) – Thyroid Stimulating Hormone (TSH) with consequent alteration of circadian rhythms of TSH has been hypothesized in some depressive disorders. Indeed, this hypothesis may explain why some forms of mood disorders were associated with anti-TPO+ or thyroid autoimmunity without hypothyroidism, as defined by routine blood tests. A slight reduction in thyroid hormone secretion such as that found in sub-clinical hypothyroidism may affect cognition and mood [12]. At variance with other tissues which mainly rely on peripherally generated Triiodothyronine, the brain utilizes preferentially circulating thyroxine directly secreted by the thyroid gland and may become hypothyroid before other organs [13]. Moreover, a study carried out in a large community sample found no association between thyroid dysfunction, including hypothyroidism defined by thyroid blood test, and the presence of depression or anxiety symptoms [14]. This survey is rather limited due to the fact that the presence of depression or anxiety symptoms was defined using a self-rating scale, whilst in the present study depression and anxiety disorders were diagnosed by means of a structured psychiatric interview according to an international classifications. However, the link between thyroid autoimmunity and depression may involve other mechanisms related to the autoimmune pathogenesis of thyroid disease rather than hypothyroidism. Since several neuroendocrine secretory systems are involved in the control of immune reaction, a common neuroendocrine dysregulation involving cytokines might concur towards the pathogenesis of both affective disorders and autoimmune disease. Recent evidence suggests that thyroid autoimmunity may be affected by the Hypothalamic-Pituitary-Adrenal axis (HPA) through the balance of proinflammatory and antiinflammatory cytokines [15]. In line with this view, the increased frequency of post-partum depression, associated to the fact that pregnancy would seem to be a "protected" period, could explain at least in part the consequences on thyroid autoimmunity elicited by HPA-related modifications to the immunitary axis. Indeed, similar phenomena are observed in rheumatoid arthritis and multiple sclerosis [16,17]. With regard to the public heath aspects of this research, should these findings be confirmed, they would constitute a most important public health issue, due to the high attributable risk found. The attributable risk is a useful measure to document the burden of risk to a community. Attributable risk depends both on the magnitude of relative risk and on the prevalence of the risk factor in the population [4]. The high attributable risk of autoimmune thyroid for Major Depressive Disorder and anxiety and depressive sub-threshold syndromes may have implication for the development of preventive interventions. Particularly, further longitudinal studies will be required to confirm whether anxiety and depressive disorders are a consequence and not a cause of thyroid autoimmunity. Limitations The potential of the study is reduced by the small sample size, particularly regard to psychiatric diagnoses less frequently observed in the general population, such as Panic Disorder; the extension of the findings is therefore rather limited. Conclusions This study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. If the findings are confirmed, they may prove to be of interest for future projects of case finding: a systematic screening for mood disorders in anti-TPO+ subjects and a systematic evaluation for thyroid diseases and thyroid autoimmunity in subjects with mood disorders may be advisable. Competing interests None declared. Authors' contributions MGC conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. AL, SM and CS participated in the statistical analysis and drafted the manuscript. MCH, SM, MC, BC, LDO participated in its design and coordination. All authors have read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Fountoulakis KN Iacovides A Grammaticos P Thyroid function in clinical subtypes of major depression BMC Psychiatry 2004 4 published on line Carta MG Hardoy MC Boi MF Mariotti S Carpiniello B Usai P Association between panic disorder, major depressive disorder and celiac disease. A possible role of thyroid autoimmunity Journal of Psychosomatic Research 2002 53 789 793 12217453 10.1016/S0022-3999(02)00328-8 Bjoro T Holmen J Kruger O Midthjell K Schreiner T Sandnes L Brochmann H Prevalence of thyroid disease, thyroid dysfunction and thyroid peroxidase antibodies in a large, unselected population. The Health study of Nord Trondelag (HUNT) Eur J Endocrinol 2000 143 639 647 11078988 Horwath E Weissman MM Tsuang MT, Tohen M, Zahner GEP Epidemiology of depression and anxiety Disorders In Textbook in Psychiatric Epidemiology 1995 New York: Wiley Liss Carta MG Kovess V Hardoy MC Morosini PL Murgia S Carpiniello B Psychiatric disorders in sardinian emigrants in Paris: a comparison with Parisians and Sardinians resident in Sardinia Social Psychiatry and Psychiatric Epidemiology 2002 37 112 117 11990007 10.1007/s001270200002 Loviselli A Oppo A Velluzzi F Independent expression of serological markers of thyroid autoimmunity and hepatitis C infection in general population J Endocrinol Invest 1999 22 660 665 10595828 Carta MG Carpiniello B Trudu MN Tarquini A Rudas N Aguglia E, Pascolo E La versione italiana della CIDI Simplified, uno studio di accuratezza e riproducibilità In Metropoli e Oltre 1994 Trieste: Tentati American Psychiatric Association Diagnostic and statistical manual of mental disorders, (DSM-IV) 1994 4 Washington DC: American Psychiatric Association Mariotti S Caturegli M Piccolo P Barbesino G Pinchera A Antithyroid peroxidase autoantibodies in thyroid diseases J Clin Endocrinol Metab 1990 71 661 669 2168432 Miettinen O Confounding and effect modification Am J Epidemiol 1974 100 350 353 Miettinen O (1974) Confounding and effect modification. Am J Epidemiol 100:350–353 4423258 Marangell B Callahan AM Mood disorders and the thyroid axis Current Opinion Psychiatry 1998 11 67 70 10.1097/00001504-199801000-00023 Haggerty JJ Prange AJ Borderline hypothyroidism and depression Annu Rev Med 1995 46 37 46 7598471 10.1146/annurev.med.46.1.37 Harris B Oretti L Lazarus J Parkes A John R Richards C Newcombe R Hall R Randomised trial of thyroxine to prevent postnatal depression in thyroid antibody positive Women Br J Psychiatry 2002 180 327 330 11925355 10.1192/bjp.180.4.327 Engum A Bjero T Myketun A Dahl AA An association between depression, anxiety and thyroid function a clinical fact or an artefact? Acta Psychiatr Scand 2002 106 27 34 12100345 10.1034/j.1600-0447.2002.01250.x Elenkov IJ Chrousos GP Stress hormones, proinflammatory and anti-inflammatory cytokines, and autoimmunity Ann Y Acad Sci 2002 966 290 303 Elenkov IJ Wilder RL Bakalov VK Dimitrov MA Fisher S Crane M Kanik KS Chrousos GP IL-12, TNF-alpha, and hormonal changes during late pregnancy and early postpartum: implications for autoimmune disease activity during these times J Clin Endocrinol Metab 2001 86 4033 4038 10.1210/jc.86.10.4933 Biondi M Zannino LG Psychological stress, neuroimmunomodulation, and susceptibility to infectious diseases in animals and man: a review Psychother Psychosom 1997 66 3 26 8996711

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BMC Psychiatry. 2004 Aug 18; 4:25

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BMC Psychiatry

10.1186/1471-244X-4-25

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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-401534166510.1186/1471-2458-4-40Research ArticleAn outbreak of Salmonella Enteritidis phage type 34a infection associated with a Chinese restaurant in Suffolk, United Kingdom Badrinath Padmanabhan [email protected] Torbjorn [email protected] Hamid [email protected] Richard [email protected] Southend Primary Care Trust & University of Cambridge, Harcourt Avenue, Southend on Sea, Essex, SS2 6HE, United Kingdom2 Suffolk Health Protection Unit, PO Box 170, South Building, St.Clements, Foxhall Road, Ipswich IP3 8LS, United Kingdom3 Department of Microbiology, Ipswich Hospital NHS Trust, Heath Road, Ipswich, Suffolk IP4 5PD, United Kingdom2004 1 9 2004 4 40 40 3 3 2004 1 9 2004 Copyright © 2004 Badrinath et al; licensee BioMed Central Ltd.2004Badrinath et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background On 30th July 2002, the Suffolk Communicable Disease Control Team received notifications of gastrointestinal illness due to Salmonella Enteritidis in subjects who had eaten food from a Chinese restaurant on 27th July. An Outbreak Control Team was formed resulting in extensive epidemiological, microbiological and environmental investigations. Methods Attempts were made to contact everybody who ate food from the restaurant on 27th July and a standard case definition was adopted. Using a pre-designed proforma information was gathered from both sick and well subjects. Food specific attack rates were calculated and two-tailed Fisher's exact test was used to test the difference between type of food consumed and the health status. Using a retrospective cohort design univariate Relative Risks and 95% Confidence Intervals were calculated for specific food items. Results Data was gathered on 52 people of whom 38 developed gastrointestinal symptoms; 16 male and 22 female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours. Food attack rates were significantly higher for egg, special and chicken fried rice. Relative risk and the Confidence interval for these food items were 1.97 (1.11–3.48), 1.56 (1.23–1.97) and 1.48 (1.20–1.83) respectively. Interviews with the chef revealed that many eggs were used in the preparation of egg-fried rice, which was left at room temperature for seven hours and was used in the preparation of the other two rice dishes. Of the 31 submitted stool specimens 28 tested positive for S Enteritidis phage type 34a and one for S Enteritidis phage type 4. Conclusion In the absence of left over food available for microbiological examination, epidemiological investigation strongly suggested the eggs used in the preparation of the egg-fried rice as the vehicle for this outbreak. This investigation highlights the importance of safe practices in cooking and handling of eggs in restaurants. ==== Body Background Infection due to Salmonella is a major public health problem in England and Wales with reports of over 14,400 infections due to Salmonella in the year 2003 [1]. The most common serotypes responsible for human infection are S Enteritidis, S Typhimurium and S Virchow [2]. Although Salmonella enterica serovar Enteritidis phage types 4, 21 and 6 have been reported in previous outbreaks [3], phage type 34a is rare and reports of outbreaks due to this serotype are scarce in the literature. Apart from one report from Wales [4], we are not aware of any other outbreaks due to S Enteritidis PT 34a infection reported in the literature from United Kingdom. In this report, we present the results of an epidemiological investigation of an outbreak due to this rare phage type associated with a Chinese restaurant in Suffolk, United Kingdom. Methods On 30th July 2002, the Suffolk Communicable Disease Control team (SCDC) was informed by the consultant microbiologist that S. Enteritidis had been isolated from stool samples of five patients. All had recently eaten a meal in a local Chinese restaurant. Further enquires revealed that there were more patients with similar food history and gastrointestinal symptoms. An Outbreak Control Team was convened on 31st July and it was decided that a full investigation should be carried out to identify the extent of the outbreak, the probable vehicle of infection and to advise on the appropriate control measures. Epidemiological The environmental health department (EHD) staff initially gathered information from people who had become ill on a standard data collection form. In the initial stages of the investigation, it became apparent that all those who became ill had eaten or had bought a take away at the restaurant on 27th July 2002. The information collected included name, address, sex, their symptoms and date of onset. The restaurant provided the list of food items that were served/sold on the day in question. This menu extended to 40 food items. This list was shown to the restaurant patrons and they were asked to state the food items they had eaten. A variety of ways was used to identify further cases including the technique of snowball sampling. This involved asking the patrons whether they were aware of any others who had similar symptoms and had eaten in the restaurant. General Practitioners providing primary care in the area were contacted and were requested to check for patients with gastrointestinal symptoms. The presenting symptoms of the patrons were diarrhoea, headache, abdominal pain and fever. The next step involved interviewing all those who had eaten/bought food on 27th July whether they became ill or not. The restaurant provided table-booking details. The following case definition was adopted for the outbreak. "Symptoms of acute gastroenteritis including one of the following: diarrhoea, vomiting or abdominal pain up to 96 hrs after having had a meal from the said restaurant including takeaway between 22 and 30 July 2002 and/or individuals who have positive stool sample for S. Enteritidis up to 96 hrs after having a meal from the restaurant including a takeaway between 22 and 30 July 2002". An analytical investigation was carried out using a retrospective cohort design. Efforts were made to identify anyone who ate or bought food at the restaurant on 27th July. Eligibility for membership of the cohort was defined as a person having the opportunity to eat any of the food items available on the day. Statistical methods Data was entered in to Statistical Package of Social Sciences version 10 [5]. Food specific attack rates and the corresponding two tailed p vales were derived by Fisher's exact test [6]. Univariate relative risk (RR) and 95% Confidence Intervals (CI) were calculated using standard cohort analysis [7]. Microbiological Stool samples were requested from all who had eaten food from the restaurant on 27th of July. Environmental sampling was not carried out as this was considered to be of limited value. There was no food left over from 27th July, but three food samples were taken on 30th July and sent for analysis to the food laboratory at Chelmsford Public Health Laboratory. Stool specimens were sent to Ipswich Hospital microbiology laboratory and were cultured for the presence of Salmonella sp. Isolates of Salmonella were forwarded to the Laboratory of Enteric Pathogens at Central Public Health Laboratory, Colindale for phage typing. Standard procedures were adopted for phage typing at the laboratory [8]. Environmental The EHD staff inspected the premises including verifying the procedures for hazard analysis and critical control point (HACCP). Egg storage and preparation of egg items were also investigated during the visit. Efforts were made to trace the egg trail back to the supplier. Results Epidemiological Data were gathered from 52 subjects who had eaten food from the restaurant on 27th July of whom 38 developed symptoms and 14 were free of symptoms. Of the 38 who became ill 16 were male and 22 were female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours suggesting a point source outbreak. Two patients received hospital care and there were no deaths. No gastrointestinal illness was reported among the kitchen staff of the restaurant in the weeks before or during the outbreak. On investigation of food preparation practices at the restaurant, it appeared dishes containing egg were the most likely vehicle for this outbreak. However, this information was not discussed when gathering data from the subjects. Data was gathered in a standardised format from all subjects to avoid any interviewer or recall bias. We had a strong "a priori" hypothesis that people who had eaten egg or food that had come in to contact with egg were at an increased risk even before looking at the data and these were analysed first. During analysis it became apparent that illness was significantly associated with the three food items that contained egg or the egg rice mixture as shown by the food specific attack rates (Table 1) and the increased relative risks (Table 2). When many other food items eaten on the day including pork-fried rice and a variety of fish dishes were analysed none of which showed an increased attack rate or was significant in the cohort analysis. Table 1 Specific attack rates of suspected foods Food item Eaten Not eaten p* value Ill Not Ill Attack rate (%) Ill Not Ill Attack rate (%) Egg fried rice 31 5 86.1 7 9 43.8 0.002 Special fried rice 13 0 100.0 25 14 64.0 0.009 Chicken fried rice 9 0 100.0 29 14 67.4 0.04 * Fisher exact test Table 2 Relative risk and 95% Confidence Intervals of suspected foods Food Item Relative Risk 95% Confidence Interval Egg fried rice 1.97 1.11 – 3.48 Special fried rice 1.56 1.23 – 1.97 Chicken fried rice 1.48 1.20 – 1.83 Microbiological A total of 31 stool specimens were submitted to the laboratory from which S. Enteritidis was isolated in 29. Twenty-eight of these 29 isolates were confirmed to be PT 34a and one as PT4. No pathogens were isolated from food samples taken from the restaurant on 30th July. Environmental During the visit to the restaurant, the EHD staff reported that there was no evidence of hazard analysis and noted many cleaning and maintenance issues. Hand wash facilities were inadequate. The chef explained that after preparing the egg rice mixture, it was left out at room temperature for the rest of the evening, and reheated when ordered. This mixture was used for some of the other fried rice items. It was estimated that on the evening of the 27th of July the egg rice mixture was left at room temperature for seven hours. The restaurant received eggs from a supplier in London every week and they were not refrigerated. Attempts to trace the egg trail were not successful. Control measures The restaurant closed voluntarily on 31st July and the EHD staff reassessed the situation on the evening of 1st of August. As they were satisfied with the arrangements, the restaurant was allowed to reopen. The owner and restaurant staff were provided with information on proper cooking methods and the importance of undertaking HACCP. Discussion The epidemiological investigation showed that eggs used in the preparation of egg-fried rice, which in turn was used in the preparation of some of the other rice items was the vehicle of infection. Isolation of the unusual phage type 34a strengthened the conclusion that eating from the restaurant was linked to a point source outbreak. In general, by the time investigations are initiated often no food material is available for laboratory analysis and the investigator has to rely on epidemiological evidence. The first step in identifying the source of an outbreak is the calculation of attack rates and the responsible food should have a significantly higher attack rate [9]. In this study, three types of food were found to have higher attack rates and were considered responsible for the outbreak (Table 1). All these items contained egg or egg rice mixture, which was left at room temperature for a long time. Cohort analysis also showed elevated RRs which were significant (Table 2). Seven sick patrons did not eat egg-fried rice. Descriptive analysis showed that all except one gave a history of eating chicken and or special fried rice. Statistical methods have better power while there is an "a priori" hypothesis as shown in the study of summer excess of leukaemia [10]. We had an "a priori" hypothesis that food items containing eggs increased the risk of illness. To our knowledge, this is the third report of an outbreak due to phage type 34a and the first of its kind in England published in the literature. In the UK, this phage type has been associated with travel abroad especially to southern Spain [11] and indigenous infections are rare. The restaurant received eggs from two sources and one of which was a packaging firm. Hence, it was not possible to determine the origin of the eggs. An outbreak due to closely related phage type 34, associated with an egg-containing dish in a Mexican restaurant in the United States has also been described [12]. There have been earlier reports of S. Enteritidis outbreaks associated with Chinese food businesses in England [13], Scotland [14] and the United States [15] although it is not clear whether any shortfalls in specific food handling techniques are responsible. In one instance [15], egg roll batter was made from pooled shelled eggs which were left at room temperature throughout the day. The proportion of eggs infected with S. Enteritidis has been reported to be low [16] and hence the risk of acquiring infection from consuming a single raw egg is much lower. However, the practice of pooling shelled eggs together with storage at room temperature as happened in our outbreak promotes bacterial multiplication and a single contaminated egg can contaminate different types of food. The role of S. Enteritidis in causing food borne outbreaks is well known as it has the ability to contaminate eggs without causing discernible illness in the birds affected [17]. Eggs have been implicated as the source of Salmonella infection in many previous outbreaks [18-22]. Hayes et al [23] in their case control study in Wales found that undercooked hens eggs are an important risk factor for sporadic Salmonella infections. We could not find any veterinary data on phage type 34 in British flocks. We searched the literature to determine whether there is any molecular relationship between phage type 34a and phage type 4. Hudson et al [24] based on the results of pulsed-field gel electrophoresis concluded that different S. Enteritidis phage types appear to be genetically related or clonal. Discussion on stability of phage types of S. Enteritidis can also be found in the literature. Conversion of phage type 4 to 24, phage type 23 to 8 and 4 to 7 have been reported. We could not find reports linking phage type 34a and phage type 4. In a recent public health investigation [25] of S. Enteritidis in raw eggshells, various serotypes of Salmonella were isolated from 23 out of 449 (5.1%) pooled samples labelled as originating from Spain. These sero/phage types included S. Enteritidis PT6a, PT5c, 13a, 14b, 58, PT6d, PT1, PT1c and PT12. A few limitations of this study are to be noted. The origin of the suspected contaminated eggs could not be traced. Although trace back exercises are key in epidemic investigations, often they are not successful due to logistic and practical reasons. In our outbreak one of the suppliers to the restaurant turned out to be a packaging firm. During our investigation, we found that there were problems with the distribution system, which prevented us from pin pointing the origin of the contaminated eggs. A possibility always exists that we missed a few subjects from this investigation and not all could be persuaded to provide a stool sample. There was no single list of all the patrons who ate/purchased food on the evening. However, all efforts were made to contact the patrons and the outbreak caused considerable publicity in the local media. Hence, we are confident that we have included most patrons. Although the precise number of patrons who were not included will never be known we are confident that their number is small and might be in the region of 10 to 15. Palmer [26] has pointed out the need to undertake outbreak investigations rapidly but at the same time with sound methodology. We tried to adopt the standard approach to investigating an outbreak including a retrospective cohort study. However, we did not attempt multivariate analysis due to the small number of subjects involved in the investigation. S. Enteritidis PT4 was isolated from one of the subjects. Further investigation revealed that this subject had recently returned from holiday in continental Europe and had suffered mild symptoms before the meal. In response to this and other outbreaks associated with eggs, a Public Health Investigation was launched in October 2002 in the UK to determine the rate of Salmonella contamination in eggs. Tests of nearly 4000 eggs showed that Salmonella was recovered from 5.3% of pooled eggs [25]. The Food Standards Agency has also produced a leaflet titled "Eggs – what caterers need to know" [27] which emphasises the importance of thoroughly cooking the eggs, buying eggs from reputable suppliers and use of pasteurised eggs when serving a vulnerable individuals. Conclusions Investigation of this outbreak was greatly facilitated by the close cooperation between local EHD, Communicable Disease Control Team, microbiological laboratory and local health care providers. Although food samples from this point source outbreak were not available for microbiological culture, epidemiological evidence pointed to eggs containing dishes as the most likely source of the outbreak. This outbreak highlights the continuing hazards of raw eggs. It is likely that the use of pasteurised eggs and the adoption of safe food preparation practices would have prevented this outbreak. Competing interests The first author (PB) served as the expert witness for the prosecution during the court proceedings. Authors' contributions PB and TS conceived and designed the study and drafted the manuscript. PB analysed the data RK oversaw the microbiological investigation. PB, TS, RK and HM all interpreted the results of the analysis and critically reviewed the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The authors would like to thank Philip Gore, Jonathan Hayes and Wai Lowe of the local environmental health department and Martin Lodge and Dalma Marossy from the food laboratory, Chelmsford Public Health Laboratory for their in put in to the investigation. We are grateful to staff of the Laboratory of Enteric Pathogens at the Central Public Health Laboratory, Colindale for carrying out the phage typing and for helpful discussions. The authors gratefully acknowledge the constructive comments and useful suggestions from both the reviewers. PB would like to thank Pat Murray, Southend Primary Care Trust for her comments on the earlier version of the document. ==== Refs CDR Weekly, Health Protection Agency CDR Weekly, Health Protection Agency CDR Weekly, Health Protection Agency Linnane E Roberts RJ Mannion PT An outbreak of Salmonella enteritidis phage type 34a infection in primary school children: the use of visual aids and food preferences to overcome recall bias in a case control study Epidemiol Infect 2002 129 35 39 12211594 10.1017/S0950268802007355 Statistical Package for Social Sciences version 10, Simple Interactive Statistical Analysis (SISA) DJR Hutchon. Calculator for Confidence interval for Relative Risk Hickman-Brenner FW Stubbs AD Farmer JJ 3rd Phage typing of Salmonella enteritidis in the United States J Clin Microbiol 1991 29 2817 2823 1757554 Tansel O Ekuklu G Otkun M Otkun MT Akata F Tugrul M A food-borne outbreak caused by Salmonella enteritidis Yonsei Med J 2003 44 198 202 12728457 Badrinath P Day NE Stockton D Seasonality in the diagnosis of acute lymphocytic leukaemia Br J Cancer 1997 75 1711 1713 9184193 Anonymous Salmonella enteritidis phage type 34 and holidays abroad Commun Dis Rep Wkly 1995 5 117 McNeil MM Sweat LB Carter SL JrWatson CB Holloway JT Manning R Altekruse SF Blake PA A Mexican restaurant-associated outbreak of Salmonella Enteritidis type 34 infections traced to a contaminated egg farm Epidemiol Infect 1999 122 209 215 10355784 10.1017/S0950268899002216 CDR weekly. Salmonella Enteritidis PT56 in Durham – final report Cowden J Hamlet N Locking M Allardice G A national outbreak of infection with Salmonella enteritidis phage types 5c and 6a associated with Chinese food businesses in Scotland, summer 2000 Epidemiol Infect 2003 130 387 393 12825722 Boyce TG Koo D Swerdlow DL Gomez TM Serrano B Nickey LN Hickman-Brenner FW Malcolm GB Griffin PM Recurrent outbreaks of Salmonella Enteritidis infections in a Texas restaurant: phage type 4 arrives in the United States Epidemiol Infect 1996 117 29 34 8760947 Centre for Disease Control and Prevention Outbreak of Salmonella enteritidis infection associated with consumption of raw shell eggs, 1991 MMWR 1992 41 369 372 1584195 Guard-Petter J The chicken, the egg and Salmonella enteritidis Environ Microbiol 2001 3 421 430 11553232 10.1046/j.1462-2920.2001.00213.x Ejidokun OO Killalea D Cooper M Holmyard S Cross A Kemp C Four linked outbreaks of Salmonella enteritidis phage type 4 infection – the continuing egg threat Commun Dis Public Health 2000 3 95 100 10902250 Trepka MJ Archer JR Altekruse SF Proctor ME Davis JP An increase in sporadic and outbreak-associated Salmonella enteritidis infections in Wisconsin: the role of eggs J Infect Dis 1999 180 1214 1219 10479150 10.1086/314984 Rejnmark L Stoustrup O Christensen I Hansen A Impact of infecting dose on severity of disease in an outbreak of food-borne Salmonella enteritidis Scand J Infect Dis 1997 29 37 40 9112296 Centre for Disease Control and Prevention Outbreaks of Salmonella serotype enteritidis infection associated with eating shell eggs – United States, 1999–2001 MMWR Morb Mortal Wkly Rep 2003 51 1149 1152 12553566 Irwin DJ Rao M Barham DW Pencheon DC Lofts C Jones PH O'Mahony M Soltanpoor N Ward LR Threlfall EJ An outbreak of infection with Salmonella enteritidis phage type 4 associated with the use of raw shell eggs Commun Dis Rep CDR Rev 1993 3 R179 R183 7507771 Hayes S Nylen G Smith R Salmon RL Palmer SR Undercooked hens eggs remain a risk factor for sporadic Salmonella enteritidis infection Commun Dis Public Health 1999 2 66 67 10462901 Hudson CR Garcia M Gast RK Maurer JJ Determination of close genetic relatedness of the major Salmonella enteritidis phage types by pulsed-field gel electrophoresis and DNA sequence analysis of several Salmonella virulence genes Avian Dis 2001 45 875 886 11785892 CDR weekly. Public Health Investigation of Salmonella Enteritidis in raw shell eggs Palmer SR Outbreak investigation: the need for 'quick and clean' epidemiology Int J Epidemiol 1995 24 S34 S38 7558549 Food Standards Agency, Eggs-What caterers need to know

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==== Front BMC Pulm MedBMC Pulmonary Medicine1471-2466BioMed Central London 1471-2466-4-61531571310.1186/1471-2466-4-6Research ArticleThe clinical significance of serum and bronchoalveolar lavage inflammatory cytokines in patients at risk for Acute Respiratory Distress Syndrome Bouros Demosthenes [email protected] Michael G [email protected] Katerina M [email protected] Panagiotis [email protected] Ioannis [email protected] Stavros [email protected] Athanasia [email protected] George [email protected] Nikolaos [email protected] Despina [email protected] Department of Pneumonology and Intensive Care Unit, University General Hospital of Alexandroupolis, Alexandroupolis, Greece2 Department of Hematology, University General Hospital of Heraklion, Heraklion, Greece3 Department of Pneumonology, University General Hospital of Heraklion, Heraklion, Greece4 Intensive Care Unit, Rethymnon General Hospital, Rethymnon, Greece5 Department of Nuclear Medicine, University General Hospital of Heraklion, Heraklion, Greece6 Department of Hematology, University General Hospital of Larissa, Larissa, Greece2004 17 8 2004 4 6 6 10 5 2004 17 8 2004 Copyright © 2004 Bouros et al; licensee BioMed Central Ltd.2004Bouros et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The predictive role of many cytokines has not been well defined in Acute Respiratory Distress Syndrome (ARDS). Methods We measured prospectively IL-4, IL-6, IL-6 receptor, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid (BALF) in 59 patients who were admitted to ICU in order to identify predictive factors for the course and outcome of ARDS. The patients were divided into three groups: those fulfilling the criteria for ARDS (n = 20, group A), those at risk for ARDS and developed ARDS within 48 hours (n = 12, group B), and those at risk for ARDS but never developed ARDS (n = 27, group C). Results An excellent negative predictive value for ARDS development was found for IL-6 in BALF and serum (100% and 95%, respectively). IL-8 in BALF and IL-8 and IL-10 serum levels were higher in non-survivors in all studied groups, and were associated with a high negative predictive value. A significant correlation was found between IL-8 and APACHE score (r = 0.60, p < 0.0001). Similarly, IL-6 and IL-6r were highly correlated with PaO2/FiO2 (r = -0.27, p < 0.05 and r = -0.55, p < 0.0001, respectively). Conclusions BALF and serum levels of the studied cytokines on admission may provide valuable information for ARDS development in patients at risk, and outcome in patients either in ARDS or in at risk for ARDS. ARDScytokinesbronchoalveolar lavageoutcome ==== Body Background Acute respiratory distress syndrome (ARDS) is characterized by respiratory failure of acute onset as a result of acute lung injury (ALI) either directly or indirectly via the blood. The main characteristics of the syndrome are diffuse inflammation and increased microvascular permeability that cause diffuses interstitial and alveolar oedema and persistent refractory hypoxemia [1]. Although a variety of insults may lead to ARDS, a common pathway may probably result in the lung damage [2-4]. A complex series of inflammatory events have been recognized during the development of ARDS but the exact sequence of the events remains unclear. Leukocyte activation and free radical release, proteases, arachidonic acid metabolites, inflammatory and anti-inflammatory cytokines results in the increased alveolar-capillary membrane permeability [5-7]. Cytokines are produced in the lung by local resident cells such as alveolar macrophages, lung epithelial cells, and fibroblasts or by cells such as neutrophils, lymphocytes, monocytes and platelets as a response to local or systemic injury [8-12]. Cytokines involved in the early phase of inflammatory response, such as IL-1, IL-2, IL-6, IL-8, [8,13,14] are secreted in response to injurious agents. Inflammatory cytokines are of critical importance in the pathophysiology of septic shock, a condition frequently leading to ARDS [15]. It has been hypothesized that the inability of lung to repair after ALI is due to a persisted inflammatory stimulus [16]. Predictive levels of inflammatory cytokines (IL-1, IL-2, IL-6, IL-8) for ARDS development in at risk patients have been reported with controversial results [5,7,11,15,16]. Cut-off values above which ARDS development occurs in at risk patients have been also reported for IL-4 and IL-10 [16]. Schutte et al [17] compared ARDS to pneumonia and cardiogenic pulmonary oedema patients and found higher IL-6 and IL-8 values in ARDS compared to remaining populations. A systematic study of the role of all main inflammatory cytokines at the same time in the pathogenesis and development of the ARDS has not been undertaken. The purpose of this study is to evaluate the role of inflammatory cytokines IL-4, IL-6, IL-6r, IL-8, and IL-10 in serum and bronchoalveolar lavage (BALF) as possible prognostic indicators for the development, severity, and outcome of patients with ARDS or at risk for ARDS. Methods Patients We studied prospectively 59 consecutive patients who were admitted in our Intensive Care Units (ICU) (Table 1). The first group (group A) included 20 patients fulfilling the criteria of ARDS [1]. All these patients were supported mechanically for their respiratory failure. The second group (group B) included 12 patients on mechanical respiratory support who had at least one condition from those suggested by Fowler et al [2] as risk factors for ARDS development. All patients in this group developed ARDS within 48 hours. The third group (group C) included 27 patients on high risk for ARDS development who never developed ARDS (Table 1). Table 1 Clinical features of the studied population on admission. Group N Sex Age (yr) Diagnosis PaO2/FiO2 APACHE II A 20 M = 14 53 ± 19 Trauma 9 121 ± 10 19.8 ± 1.4 F = 6 Pneumonia 3 Sepsis 2 Transfusion 2 Pancreatitis 2 Intoxication 1 Burns 1 B 12 M = 8 56 ± 20 Sepsis 3 239 ± 30 20.5 ± 1.3 F = 4 Pneumonia 4 Trauma 4 Pancreatitis 1 C 27 M = 21 49 ± 18 Sepsis 12 276 ± 16 16.0 ± 1.1* F = 6 Trauma 5 Pneumonia 2 Transfusion 2 Intoxication 2 Arrest 2 Pancreatitis 2 D 33 M = 20 36 ± 16 F = 13 M = male, F = female, burns = >40% of the body surface. * p < 0.05 group A vs groups B and C. For patients' classification, the following criteria were employed: 1. The ARDS criteria of the American-European Consensus Conference on ARDS (1): a. acute onset, b. bilateral chest radiographic infiltrates, c. pulmonary artery occlusion pressure of ≤18 mm Hg, or no evidence of left atrial hypertension, and d. impaired oxygenation regardless of the PEEP concentration, with a PaO2/FiO2 ratio of ≤ 300 torr for ALI and ≤ 200 torr for ARDS. 2. The high-risk criteria for ARDS development according to Fowler et al [2]). 3. The criteria for pneumonia according to EPIC study [18], and 4. The criteria for septic syndrome according to Bone et al [19]. Acute Physiology and Chronic Health Evaluation-II (APACHE II) scoring system was used for grading the disease severity [20]). The main clinical features of the patients are shown in Table 1. The protocol was approved by the Ethics Committee of our institutions. After admission to the ICU blood samples were obtained from a central venous line within 2 hours. APACHE II score and PaO2/FiO2 values were obtained at the time of sample collection. The blood was collected in a heparinized vacutainer tube and kept immediately at 4°C. After centrifugation at 1500 g at 4°C, the plasma was kept at -80°C until the measurement. Immediately after blood collection BALF was obtained by fiberoptic bronchoscopy. The fluid was filtered through nylon net to remove the mucous secretions, and centrifuged at 500 g for 10 min to remove cells. The supernatant was kept in cryotubes at -80°C in aliquots of 0.5 ml. The method of micro-lavage was used as described previously [21]. The following criteria were used for an acceptable sample: a. The procedure should be shorter than 1 min, while the time of saline staying into the lungs should be less than 20 sec, b. recovery of more than 50% of the saline used for the lavage, c. absence of obvious blood contamination in the BALF, and d. the level of urea in the BALF should be more than 0.4 mmol. The urea level was used as an index of BALF dilution [21]. To check the accuracy of the method, two subsequent lavages were taken in 8 patients and all the studied parameters in the two samples did not differ significantly. Measurement of the plasma cytokines The assay method for cytokine measurement was the same for blood and BALF samples. Determination of plasma cytokines was done with solid phase enzyme-linked immunosorbent assay (ELISA) methodology based on the quantitative immunometric sandwich enzyme immunoassay technique [22]. Reagents for the studied cytokines were obtained from several sources (kits of R&D systems, Inc. Minneapolis, MN, USA, for IL-6R, kits of Genzyme Diagnostics, Cambridge, MA, USA for IL-8, IL-10, and RIA kits of Amersham, Buckinghamshire, UK, for IL-4, IL-6) were used according to manufacturer's instructions. Intra-assay and inter-assay reproducibility was checked and found more than 90%. To calculate the dilution factor of the BALF, urea values in the plasma and BALF were used because this low molecular weight substance is found to be in the body fluids at the same concentration as in the blood. Statistical analysis Data analysis was carried out using SPSS 8.0 statistical software (SPSS Inc., Chicago, IL). Results are expressed as mean ± 1SD, or median (range), unless otherwise indicated. The Mann-Whitney non-parametric test was used to compare the mean values of the cytokines in the blood and BALF in the various groups. Receiver-operating characteristic (ROC) correlation was used to find the optimal cut-off values of the studied cytokines for ARDS development in patient at risk and survival of the patient population [23]. For tests of association, we calculated Spearman's correlation coefficient. A p value <0.05 was considered to be statistically significant. Results There was no significant difference in the mean age of the patients among the three studied groups. Using APACHE-II score to determine the severity of the disease, significant difference between group A and group C (p = 0.04) and group B and C (p = 0.045) was found, and not between groups A and B (p = 0.06). The mean time of staying in the ICU did not differ among the three groups. Predictive capabilities of BALF mediators for onset of ARDS The mean values (+/- SD) of the measured cytokines in BALF and serum in the three studied patient groups are shown in Table 2. A significant difference was found for BALF IL-6r, which was higher in group A than in groups B and C (p < 0.0001). Similarly, BALF IL-6 was higher in groups A and B compared to C (p < 0.01). Table 2 Mean (+/-SD) BALF and serum levels of studied cytokines in the three groups of patients. Group A (n = 20) Group B (n = 12) Group C (n = 27) Cytokines (pg/mL) BALF Serum BALF Serum BALF Serum IL-4 260 ± 181 158 ± 68 284 ± 119 95 ± 351 242 ± 147 83 ± 681 IL-6 538 ± 4322 388 ± 3243 1135 ± 13822 505 ± 217 318 ± 446 313 ± 3733 IL-6r 180 ± 52 30 ± 25 80 ± 374 34 ± 26 73 ± 224 39 ± 45 IL-8 480 ± 222 3525 ± 15235 492 ± 165 3543 ± 22655 467 ± 179 2553 ± 2824 IL-10 62 ± 24 117 ± 60 111 ± 98 177 ± 117 73 ± 50 118 ± 84 1p < 0.0001 versus group A, 2p < 0.01 versus group C. 3p < 0.05 versus group B. 4p < 0.0001 versus group A. 5p < 0.0001 versus group C. Predictive capabilities of serum mediators for onset of ARDS Serum levels of IL-4 were higher in group A compared to groups B and C (p < 0.0001). Serum IL-6 was higher in group B compared to group A and C (p < 0.05). Serum levels of IL-8 was higher in group A and group B compared to group C (p < 0.0001) (Table 2). Predictive values for ARDS development in at risk patients (groups B and C) for BALF and serum IL-6 are shown in Table 3. IL-6 negative predictive values for ARDS development were 100% and 95% for BALF cut off value of 195 pg/ml and serum cut off value of 255 pg/ml, respectively. Table 3 BALF and serum IL-6 predictive values for ARDS development in patients at risk (n = 39, groups B+C). Criterion PPV NPV Sensitivity Specificity Prevalence 95% CI BALF IL-6, (pg/mL) >195 44 100 100 62 24 0.62–0.91 Serum IL-6, (pg/mL) >255 44 95 88 65 24 0.60–0.90 PPV: positive predictive value, NPV: negative predictive value. CI: confidence interval Predictive capabilities of BALF mediators for survival of ARDS Mean (SEM) values in BALF and serum of the studied mediators in the survivors and non-survivors (groups A+B+C) are shown in Table 4. BALF levels of IL-6, IL-6r and IL-8 were significantly higher in those who did not survive (p < 0.05, p < 0.05 and p < 0.0001, respectively). Table 4 Mean (SEM) BALF and serum levels of the measured cytokines in all patients (Groups A+B+C) according to survival. BALF Serum Cytokines (pg/ml) survivors (n = 30) non-survivors (n = 29) survivors (n = 30) non-survivors (n = 29) IL-4 262 ± 188 247 ± 108 72 ± 51 154 ± 68*** IL-6 313 ± 427 743 ± 877* 218 ± 191 530 ± 389*** IL-6r 94 ± 52 129 ± 69* 18 ± 19 53 ± 42** IL-8 340 ± 109 621 ± 144*** 1269 ± 830 4957 ± 1965*** IL-10 69 ± 37 82 ± 70 70 ± 16 188 ± 84*** * p < 0.05, ** p < 0.001, *** p < 0.0001 versus survivors group. Patients with ARDS (group A) who did not survive had significantly higher BALF levels of IL-8 (p < 0.0001) and significantly lower BALF levels of IL-10 (p < 0.001) (Table 5). Patients at risk (groups B and C) who did not survive had significantly higher BALF levels of IL-8 (p < 0.0001) (Table 6). IL-6, IL6-r and IL-8 BALF concentration cut off predictive values for surviving patients are shown in Table 7. BALF IL-8 was also elevated in patients of group C who died (p < 0.0001) (Table 8). Table 5 Mean (SEM) BALF and serum levels of cytokines in ARDS patients according to survival (group A). BALF Serum Cytokines (pg/ml) Survivors (n = 6) Non-survivors (n = 14) Survivors (n = 6) Non-survivors (n = 14) IL-4 352 ± 291 224 ± 115 155 ± 63 160 ± 73 IL-6 361 ± 238 606 ± 476 213 ± 140 455 ± 353 IL-6r 180 ± 38 180 ± 59 24 ± 24 31 ± 25 IL-8 218 ± 79 581 ± 167*** 2028 ± 700 4100 ± 1353* IL-10 80 ± 12 55 ± 24** 63 ± 11 138 ± 58* *p < 0.05, ** p < 0.001, *** p < 0.0001 versus survivors group. Table 6 Mean (SEM) BALF and serum levels of cytokines in at risk patients who developed or not ARDS according to survival (Groups B and C). BALF Serum Cytokines (pg/ml) Survivors (n = 24) Non-survivors (n = 15) Survivors (n = 24) Non-survivors (n = 15) IL-4 242 ± 160 271 ± 100 53 ± 21 147 ± 65*** IL-6 302 ± 463 890 ± 1178 219 ± 203 612 ± 423** IL-6r 74 ± 29 74 ± 18 17 ± 18 77 ± 44*** IL-8 368 ± 96 664 ± 105*** 1097 ± 770 5885 ± 2151*** IL-10 67 ± 40 111 ± 91 72 ± 17 243 ± 74*** * p < 0.01, ** p < 0.001, *** p < 0.0001 versus survivors group. Table 7 Predictive BALF and serum levels (pg/ml) for surviving patients of all groups (n = 59) Criterion PPV NPV Sensitivity Specificity 95 % CI BALF IL-6 299 68 70 68 70 0.57–0.83 IL-6r 101 65 63 52 74 0.52–0.79 IL-8 481 96 90 88 96 0.85–0.99 Serum IL-4 84 81 100 100 78 0.77–0.96 IL-6 160 69 94 96 59 0.69–0.92 IL-6r 18 76 78 76 78 0.66–0.89 IL-8 2340 92 96 96 93 0.90–0.99 IL-10 98 96 93 92 96 0.84–0.99 PPV: positive predictive value, NPV: negative predictive value, CI: confidence interval. Table 8 Mean (SEM) BALF and serum levels of cytokines in at risk patients who did not develop ARDS (group C) BALF Serum Cytokines (pg/ml) Survivors (n = 19) Non-survivors (n = 8) Survivors (n = 19) Non-survivors (n = 8) IL-4 242 ± 169 243 ± 69 51 ± 22 169 ± 78** IL-6 297 ± 497 374 ± 285 199 ± 197 620 ± 559* IL-6r 70 ± 25 80 ± 7 17 ± 19 98 ± 43** IL-8 377 ± 95 711 ± 102*** 1102 ± 810 6496 ± 2543*** IL-10 69 ± 43 85 ± 69 73 ± 17 240 ± 70*** *p < 0.05, **p < 0.001, ***p < 0.0001 versus survivors group. Predictive capabilities of serum mediators for survival of ARDS Cytokine concentration cut off predictive values for surviving patients are shown in Table 7. All studied mediators were found at higher levels in the serum of non-survivors (p < 0.001 to p < 0.0001). In patients at risk (groups B and C) who did not survive all serum mediators were significantly elevated (p < 0.001 to p < 0.0001) (Table 6). Serum levels of all the studied molecules were increased in all patients that did not survive (p < 0.05 to p < 0.0001) (Table 8). In survivors BALF/serum ratios were significantly higher for IL-4, IL-8, IL-10 (p < 0.0001, p < 0.001 and p < 0.0001, respectively), due to lower serum levels and not to higher BALF levels. Correlations of the studied cytokines Furthermore, the serum levels of all studied mediators were significantly correlated to APACHE II score. Serum IL-8 exhibited the strongest correlation with APACHE II score (Figure 1). The level of IL-8 in the BALF were found to be significantly correlated to APACHE II score (r = 0.60, p < 0.0001). Figure 1 Positive strong correlation of serum levels of IL-8 to APACHE II score (Spearman's rank order correlation coefficient). PaO2/FiO2 ratio was significantly correlated to the BALF levels of IL-6 and IL-6r (r = -0.27, p < 0.05; r = -0.55, p < 0.0001; respectively) (Figure 2) and to serum levels of IL-4 (r = -0.36, p < 0.05). Figure 2 Negative correlation of BALF levels of IL-6, and IL6 receptor to PaO2/FiO2 ratio (Spearman's rank order correlation coefficient). Discussion We designed this study in order to explore factors that could have prognostic value for the development, the severity, and the outcome of patients with ARDS and at risk for ARDS. Prediction of ARDS development We observed that BALF levels of IL-6r were significantly higher in group A than in groups B and C (p < 0.0001), while no difference was observed in serum levels among the three groups of patients. Interestingly, the BALF and serum levels of cytokine IL-6 were significantly higher in patients at risk who developed ARDS (group B) compared to the other two groups. This observation differs from previous studies [24,25], probably reflecting the different patient population from our study. However, our results are in agreement with previous reports regarding the luck of its prediction capacity for ARDS onset [25,26], since both BALF and serum IL-6 levels, showed a low positive predictive value according to the ROC analysis. Patients of group A and group B had higher serum levels of the inflammatory cytokine IL-8 compared to the patients of group C, but neither serum nor BALF IL-8 levels were predictive for ARDS development. In two studies, Miller et al, [27] found that IL-8 in BAL at the beginning of ARDS was highest in patients who died, and Donnelly et al [28] found that IL-8 was highest in patients at risk for ARDS who later developed ARDS. Unfortunately, subsequent studies have found that IL-8 does not predict outcome either at the outset or during the course of ARDS [5]). Meduri et al [16] found that all cytokines measured remained high during the course of ARDS in patients who died. The importance of considering anti-inflammatory constituents of BALF is shown by Donnelly et al [28] who found that patients with ARDS who died had significantly lower initial concentrations of IL-10 in BAL than patients who lived. Parson et al [29]) studied serial levels of IL-1ra and IL-10 in patients who were identified as being at risk for the development of ARDS. Initial IL-1ra levels were significantly higher (p < 0.0001) in the patients than in normal control subjects. Similarly, IL-10 levels were increased in patients compared with normal control subjects but did not predict the development of ARDS. Like IL-1ra levels, initial IL-10 levels were significantly higher (p = 0.005) in patients who died compared with survivors. However, in other studies increased levels of IL-4, and IL-10 in serum and/or BALF were found to have beneficial effect in pre-ARDS patients [13,14]. Thus, the heterogeneity of patients in the various studies may be a reason for the contradictive results reported earlier. Prediction of outcome Patients who died had significantly higher levels of IL-6, IL-6r and IL-8 in BALF than those who finally survived, while all mediators studied were significantly higher in the serum of non-survivors. The rationale for analysis of cytokine concentrations in BAL fluid is that inflammatory cytokines, like IL-6 and IL-8 are known to be produced by airway epithelial cells and activated pulmonary macrophages in response to a variety of infectious agents and other triggers of airway inflammation [30]. During ARDS, the alveolar epithelial-endothelial barrier is disrupted, and cytokines produced in the lung are released into the systemic circulation. This is believed to be a potential mechanism for the development of systemic inflammatory response syndrome [31,32]. The relationship between circulatory and pulmonary cytokines levels and outcome provides support to the hypothesis that poor outcome in ARDS is related to a persistent inflammatory process [30-33]. In addition, in agreement with our findings, bronchoalveolar concentrations of the above cytokines have been reported to be increased in patients with or at risk for ARDS [33]. As demonstrated by Meduri and co-workers, BAL fluid concentrations of IL-8 and IL-6 were significantly higher in nonsurvivors than in survivors [31]. Increased BAL levels most likely indicate intrapulmonary overproduction and not increased permeability [33]. Therefore, determination of these selected inflammatory cytokines in BAL fluid in ARDS could be of prognostic relevance [33,34]. Regarding serum levels, patients at risk (groups B and C) who died had all molecules significantly increased (Table 6), suggesting that systemic inflammatory over-response in critically ill patients may be destructive leading to multiple organ dysfunction and poor outcome. Serum levels of all the studied molecules were increased taking all patients together (groups A+B+C, Table 4) or separate (Tables 5, 6, and 8) that did not survive, suggesting that cytokinemia might reflect the severity and extension of inflammation but is not the only factor related to ARDS development. Interestingly, only IL-8 and IL-10 both in BALF and serum were higher in ARDS patients who died. These results are consistent with those of Donnelly and coworkers, who found elevated concentrations of IL-10 in BALF of 28 patients with ARDS [35]. However, our results differ from those of Armstrong and Millar, who found significantly lower concentrations of IL-10 in a small number group of patients at risk for ARDS [36]. In addition, low concentrations of IL-10 in BALF from patients with ARDS were found to be associated with increased mortality [35,37]. In contrast, all cytokines were elevated in those who died taking together all the patients at risk (groups B and C). Regarding the survival prediction, IL-8 and IL-10 showed the higher serum positive predictive value (92 and 96%, respectively), while IL-4 had the higher serum negative predictive value and sensitivity, taking together all patients. Relation to severity of lung injury Regarding the relation of the studied molecules and the severity of lung injury, a negative correlation was found between BALF IL-6, and IL-6r and PaO2/FiO2. The same was true for serum IL-4 and PaO2/FiO2. All the studied molecules in the serum were positively correlated with the APACHE II score, as was BALF IL-8. It is probable that this cytokine is closely related to the extension of tissue damage and organ failure. Conclusions In conclusion, our data show that the predictive role of most of the studied molecules both in serum and BALF for ARDS development is valuable. In addition, almost all of them are good predictors of outcome in these patients. Further studies with greater number of patients with various subgroups of ARDS as well as stricter grouping criteria should be designed to investigate the complex network of these molecules and their receptors in ARDS and their value as predictive factors in these patients. Competing interest None declared. Authors' contributions DB conceived of the study, and participated in its design and coordination and drafted the manuscript MGA participated in the design of the study, carried out immunoassays and drafted the manuscript KMA carried out immunoassays and drafted the manuscript PA patients data and samples collection IP patients data and samples collection SA patients data and samples collection GP Performed statistical analysis AP patients data and samples collection NK carried out RIA measurements DP KMA carried out immunoassays and drafted the manuscript All authors read and approved the final manuscript Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments We thank Konstantinos Perisynakis for assisting in statistical analysis. ==== Refs Bernard GR Artigas A Brigham KL Carlet J Falke K Hudson L Lamy M Legall JR Morris A Spragg R The American-European Consensus Conference onARDS Definitions, Mechanisms, Relevant Outcomes, and Clinical Trial Coordination Am J Respir Crit Care Med 1994 149 818 824 7509706 Fowler AA Hamman RF Good JT Benson KN Baird M Eberle DJ Petty TL Hyers TM Adult respiratory distress syndrome risk with common predispositions Ann Intern Med 1983 98 593 597 6846973 Rinaldo JE. Christman JW Mechanisms and Mediators of the adult respiratory distress syndrome Clin Chest Med 1990 11 621 629 2268993 Pepe PE Potkin RT Reus DH Hudson LD Carrico CJ Clinical predictors of the adult respiratory distress syndrome Am J Surg 1982 144 124 128 7091520 10.1016/0002-9610(82)90612-2 Goodman RB Strieter RM Martin DP Steinberg KP Milberg JA Maunder RJ Kunkel SL Walz A Hudson LD Martin TR Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome Am J Respir Crit Care Med 1996 154 602 611 8810593 Bellingan GJ The pathogenesis of ALI/ARDS Thorax 2002 57 540 6 12037231 10.1136/thorax.57.6.540 Tomashefski JF Jr Pulmonary pathology of acute respiratory distress syndrome Clin Chest Med 2000 21 435 466 11019719 Meduri GU Kanangat S Stefan J Tolley E Schaberg D Cytokines IL-1beta, IL-6, and TNF-alpha enhance in vitro growth of bacteria Am J Respir Crit Care Med 1999 160 961 967 10471625 Park WY Goodman RB Steinberg KP Ruzinski JT Radella F 2ndPark DR Pugin J Skerrett SJ Hudson LD Martin TR Cytokine balance in the lungs of patients with acute respiratory distress syndrome Am J Respir Crit Care Med 2001 164 1896 1903 11734443 Tamura DY Moore EE Partrick DA Johnson JL Zallen G Silliman CC IL-6 augments neutrophil cytotoxic potential via selective enhancement of elastase release J Surg Res 1998 76 91 94 9695746 10.1006/jsre.1998.5295 Kiehl MG Ostermann H Thomas M Muller C Cassens U Kienast J Inflammatory mediators in bronchoalveolar lavage fluid and plasma in leukocytopenic patients with septic shock-induced acute respiratory distress syndrome Crit Care Med 1998 26 1194 1199 9671368 10.1097/00003246-199807000-00019 Martin TR Lung cytokines and ARDS: Roger S. Mitchell Lecture Chest 1999 116 2S 8S 10424558 10.1378/chest.116.suppl_1.2S Mulligan SM Jones ML Vaporciyan AA Maureen CH Ward PA Protective effects of IL-4 and IL-10 against immune complex-induced lung injury J Immunol 1993 151 5666 5674 7901280 Van Laetherm JL Eskinazi R Louis H Rickaert F Robberecht P Devieve J Multisystem production of interleukin 10 limits the severity of acute pancreatitis in mice Gut 1998 43 408 413 9863488 Headley AS Tolley E Meduri GU Infections and the inflammatory response in acute respiratory distress syndrome Chest 1997 111 1306 21 9149588 Meduri GU Kohler G Headley S Tolley E Stentz F Postlethwaite A Inflammatory cytokines in the BAL of patients with ARDS. Persistent elevation over time predicts poor outcome Chest 1995 108 1303 14 7587434 Schutte H Lohmeyer J Rosseau S Ziegler S Siebert C Kielisch H Pralle H Grimminger F Morr H Seeger W Bronchoalveolar and systemic cytokine profiles in patients with ARDS, severe pneumonia and cardiogenic pulmonary oedema Eur Respir J 1996 9 1858 67 8880103 10.1183/09031936.96.09091858 Vincent JL Bihari DJ Suter PM Bruining HA White J Nicolas-Chanoin MH Wolff M Spencer RC Hemmer M Prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) study. EPIC International Advisory Committee JAMA 1995 274 639 644 7637145 10.1001/jama.274.8.639 Bone RC Balk RA Cerra FB Dellinger RP Fein AM Knaus WA Schein RM Sibbald WJ The ACCP/SCCM Consensus Conference Committee: American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference:Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis Chest 1992 101 1644 1655 1303622 Knaus WA Draper EA Wagner DP Zimmerman JE APACHE II: a severity of disease classification system Crit Care Med 1985 13 818 29 3928249 Baldwin DR Wise R Andrews JM Honeybourne D Microlavage: a technique for determining the volume of epithelial lining fluid Thorax 1991 46 658 662 1948795 Grassi J Frobert Y Pradelles P Chercuitte F Gruaz D Dayer JM Poubelle PE Production of monoclonal antibodies against IL-1a and IL-1b: development of the two-enzyme immunometric assays (EIA) using acetylcholinesterase and their application to biological media J Immunol Meth 1989 123 193 210 10.1016/0022-1759(89)90223-8 Zweig MH Campbell G Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine Clin Chem 1993 39 561 77 8472349 Mitchell RS Lung cytokines and ARDS Chest 1999 116 2S 8S 10424558 10.1378/chest.116.suppl_1.2S Meduri GU Headley S Kohler G Stentz F Tolley E Umberger R Leeper K Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time Chest 1995 107 1062 1073 7705118 Calandra T Gerain J Heumann D Baumgartner JD Glauser MP High circulating levels of interleukin-6 in patients with septic shock: evolution during sepsis, prognostic value, and interplay with other cytokines. The Swiss-Dutch J5 Immunoglobulin Study Group Am J Med 1991 91 23 29 1907101 10.1016/0002-9343(91)90069-A Miller EJ Cohen AB Nagao S Griffith D Maunder RJ Martin TR Weiner-Kronish JP Sticherling M Christophers E Matthay MA Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality Am Rev Respir Dis 1992 146 427 432 1489135 Donnelly SC Strieter RM Reid PT Kunkel SL Burdick MD Armstrong I Mackenzie A Haslett C The association between mortality rates and decreased concentrations of interleukin-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome Ann Intern Med 1996 125 191 196 8686976 Parsons PE Moss M Vannice JL Moore EE Moore FA Repine JE Circulating IL-1ra and IL-10 levels are increased but do not predict the development of acute respiratory distress syndrome in at risk patients Am J Respir Crit Care Med 1997 155 1469 1473 9105096 Shelhamer JH Levine SJ Wu T Jacoby DB Kaliner MA Rennard SI Airway inflammation Ann Intern Med 1995 123 288 304 7611596 Agouridakis P Kyriakou D Alexandrakis MG Prekates A Perisinakis K Karkavitsas N Bouros D The predictive role of serum and bronchoalveolar lavage cytokines and adhesion molecules for acute respiratory distress syndrome development and outcome Respir Res 2002 23 25 12537603 10.1186/rr193 Agouridakis P Kyriakou D Alexandrakis MG Persinakis K Karkavitsas N Bouros D Association between increased levels of IL-2 and IL-15 and outcome in patients with early acute respiratory distress syndrome Eur J Clin Invest 2002 32 862 867 12423329 10.1046/j.1365-2362.2002.01081.x Meduri GU Kohler G Headley S Tolley E Stentz F Postlethwaite A Inflammatory cytokines in the BAL of patients with ARDS Chest 1995 108 1303 1314 7587434 Kiehl MG Ostermann H Thomas M Muller T Cassens U Kienast J Inflammatory mediators in bronchoalveolar lavage fluid and plasma in leukocytopenic patients with septic shock-induced acite respiratory distress syndrome Crit Care Med 1998 26 1194 1198 9671368 10.1097/00003246-199807000-00019 Donnelly SC Strieter RM Reid PT Kunkel SL Burdick MD Armstrong I Mackenzie A Haslett C The association between mortality rates and decreased concentrations of IL-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome Ann Intern Med 1996 125 191 196 8686976 Armstrong L Millar AB Relative production of tumour necrosis factor alpha and interleukin in adult respiratory distress syndrome Thorax 1997 52 442 446 9176536 Park WY Goodman RB Steinberg KP Ruzinski JT Radella F 2ndPark DR Pugin J Skerrett SJ Hudson LD Martin TR Cytokine balance in the lungs of patients with acute respiratory distress syndrome Am J Respir Crit Care Med 2001 164 1896 1903 11734443

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==== Front BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-4-211532769410.1186/1472-6963-4-21Study ProtocolEncouraging physician appropriate prescribing of non-steroidal anti-inflammatory therapies: protocol of a randomized controlled trial [ISRCTN43532635] Doupe Malcolm [email protected] Alan [email protected] Brent [email protected] Lori-Jean [email protected] Colleen [email protected] Glen TD [email protected] Laura [email protected] Kat [email protected] Primary Health Care Research Unit, St Boniface Research Centre, Winnipeg, Canada2 Department of Family Medicine, University of Manitoba, Winnipeg, Canada3 Department of Continuing Medical Education, University of Manitoba, Winnipeg, Canada4 Department of Patient Health, Merck Frosst Canada Ltd., Kirkland, Canada5 Faculty of Pharmacy, University of Manitoba, Winnipeg, Canada6 CIADS Research, Centre for Inflammatory and Arthritic Disease Studies, Winnipeg, Canada2004 24 8 2004 4 21 21 25 3 2004 24 8 2004 Copyright © 2004 Doupe et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are a widely used class of therapy in the treatment of chronic pain and inflammation. The drugs are effective and can be relatively inexpensive thanks to available generic versions. Unfortunately the traditional NSAIDs are associated with gastrointestinal complications in a small proportion of patients, requiring costly co-therapy with gastro-protective agents. Recently, a new class of non-steroidal anti-inflammatory agents known as coxibs has become available, fashioned to be safer than the traditional NSAIDs but priced considerably higher than the traditional generics. To help physicians choose appropriately and cost-effectively from the expanded number of anti-inflammatory therapies, scientific bodies have issued clinical practice guidelines and third party payers have published restricted reimbursement policies. The objective of this study is to determine whether an educational intervention can prompt physicians to adjust their prescribing in accordance with these expert recommendations. Methods This is an ongoing, randomized controlled trial. All primary care physicians in Manitoba, Canada have been randomly assigned to a control group or an intervention study group. The educational intervention being evaluated consists of an audit and feedback mechanism combined with optional participation in a Continuing Medical Education interactive workshop. The primary outcome of the study is the change, from pre-to post-intervention, in physicians' appropriate prescribing of non-steroidal anti-inflammatory therapies for patients requiring chronic treatment. Three classes of non-steroidal anti-inflammatory therapies have been identified: coxib therapy, traditional NSAID monotherapy, and traditional NSAID therapy combined with gastro-protective agents. Appropriate prescribing is defined based on international clinical practice guidelines and the provincial drug reimbursement policy in Manitoba. ==== Body Background Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are a widely prescribed class of therapy used to relieve pain and inflammation. The drugs have been shown to be effective for a variety of common disorders (hence their widespread use), most notably chronic osteoarthritis and rheumatoid arthritis. They are relatively inexpensive due to the available generic versions, but unfortunately have clinically important drawbacks related to their gastrointestinal (GI) toxicity [1]. Each year, about 1% to 1.5% of patients taking traditional NSAIDs experience serious GI side effects such as perforations, ulcers, and bleeding [2-5]. When multiplied by the total number of NSAID users this translates into significant patient morbidity and mortality [1,6] and is associated with considerable health care costs related to hospitalizations or to the prescribing of expensive gastro-protective agents (GPAs) [7-12]. The cause of this GI toxicity is the "non-selective nature" of traditional NSAIDs that block both cyclo-oxygenase-2 (Cox-2), an enzyme involved in the production of inflammation and pain, and Cox-1, a related molecule that functions in GI tract mucosal protection and platelet function [13]. In the last five years, a new class of non-steroidal anti-inflammatory agents has become available to physicians, fashioned specifically to be safer than the traditional NSAIDs but priced at least two to three times higher than the generic versions of traditional NSAIDs [14]. The new drugs preferentially inhibit Cox-2 enzymes as compared to COX-1 molecules, and therefore have been christened "Cox-2 selective inhibitors" or "coxibs" for short. Large clinical trials comparing the use of coxibs to traditional NSAIDs have lent support to the concept that the new agents offer an improved GI safety profile while maintaining comparable analgesic efficacy in patients with chronic arthritis [3,4,15]. The trials, however, have also hinted that the improved GI safety may be compromised by concomitant use of low-dose aspirin [4] and may come at the expense of some cardiovascular safety, although these data remain controversial [16,17]. Regardless, the introduction of these new anti-inflammatory agents has prompted the question: when is it appropriate and cost-effective to prescribe coxibs versus traditional NSAID monotherapy or traditional NSAIDs in combination with GPAs? To assist physicians in selecting from the different classes of anti-inflammatory agents, various scientific bodies have published clinical guidelines and third party payers have issued restricted reimbursement criteria [18-24]. These guidelines and reimbursement criteria in general propose that, for the treatment of chronic osteoarthritis and rheumatoid arthritis, coxibs should be used in lieu of traditional NSAID monotherapy when patients have an elevated risk for serious GI events. Traditional NSAID therapy in combination with certain GPAs (misoprostol or proton pump inhibitors) is also recommended as an alternative for most high-risk patients. High-risk patients are identified as individuals who have one or more of the following clinical characteristics: a history of peptic ulcer disease; advanced age (over 65 years); concomitant use of corticosteroids or anticoagulants; multiple comorbid conditions; or use of high doses or multiple NSAIDs [1,5,6,25,26]. These high-risk patients are considered to benefit from the improved GI safety of coxibs or from the GI protection afforded by GPAs. Furthermore, coxibs and GPA co-therapy have been shown to be cost-effective in such high-risk patients, as the increased cost of the drugs is partially offset by significant reductions in morbidity and mortality and related expenses [14,27-29]. With these guidelines and policies on prescribing in place, it is now important for physicians to adjust their prescribing practices accordingly. This is true both for rheumatology specialists and for physicians in the primary care setting where osteoarthritis patients are frequently managed. Unfortunately, experience and educational research show that simply making guidelines available does not elicit behavioural change from physicians [30-33]. In the case of anti-inflammatory drug prescribing, there is already evidence showing that despite the availability of guidelines and reimbursement policies, physicians' choices of drugs remain suboptimal both in terms of the inappropriate use of traditional NSAIDs and the non-cost-effective use of coxibs [34-38]. The current use of anti-inflammatory drugs suggests a need for strategies that will prompt physicians to change their prescribing practice in accordance with the expert recommendations. Preferably, strategies should be investigated that have proved successful at altering physician behaviour in other settings. In this paper, we describe a randomized controlled study, which we are presently conducting, to evaluate the impact of an educational intervention on primary care physicians' prescribing of non-steroidal anti-inflammatory therapies. The study, being conducted in the province of Manitoba, Canada, tests the hypothesis that this intervention will significantly improve physician appropriate prescribing of anti-inflammatory drugs in compliance with international clinical practice guidelines and Manitoba's restricted drug reimbursement policy [18-22]. The intervention being tested is modelled on proven approaches to changing physician behaviour; it consists of an audit and feedback mechanism with optional participation in a Continuing Medical Education (CME) interactive workshop. This manuscript summarizes Phases II to V of a larger initiative entitled the Manitoba Appropriate Anti-Inflammatory Utilization Initiative (MAAUI). The study began in November 2000 and is currently in the data analysis stage. In this paper, we relate in detail the protocol of this randomized controlled study. Methods Study population MAAUI is a province-wide population-based study. All primary care physicians (non-specialists) registered with the College of Physicians and Surgeons of Manitoba and practicing in Manitoba since July 1995 were eligible to enter MAAUI. The study excluded residents, new physician graduates, physicians registered as specialists, and physicians who started practicing in the province of Manitoba after July 1995. The eligible study population thus totalled approximately 884 physicians. All eligible physicians were automatically entered into the MAAUI protocol. Physicians who were randomized to intervention group of the research were given the option to withdraw from the study. Six family physicians who agreed to act as facilitators for the MAAUI CME workshop were not included in the study population. Study design The MAAUI study design is depicted in Figure 1. Primary care physicians were allocated to the control and intervention arms of the study using a stratified randomization process. Specifically, study participants were divided into groups according to the geographical area of their practice within Manitoba. These groups were stratified by physicians' "urban" (within Winnipeg – the provincial capital, population size 676,700) or "rural" (outside of Winnipeg, population size 468,300) practice location. Randomization within each stratum was then carried out at the level of the group. The MAAUI research team originally identified 12 urban and 11 rural areas in Manitoba based on existing community boundaries within Winnipeg and the presence of distinct regional health districts outside of Winnipeg. Because one urban and rural area each contained very few primary care physicians (less than five), these areas were joined to neighbouring regions resulting in 11 urban and 10 rural groups. Urban and rural groups were randomized in such a way as to achieve a 1:2 ratio of control to intervention groups. Four of the urban groups and three of the rural groups were randomized to the control arm, and the remaining seven urban and seven rural groups were randomized to the intervention arm. This study design was chosen for several reasons. First, it offered the scientific rigour that is associated with randomization and the use of a control group. It also ensured that both rural and urban Manitoban physician groups had an equal opportunity to take part in and learn from the study intervention. The study design was also particularly amenable to testing an intervention that required physicians to congregate in common locations (i.e. for the CME workshop). The 1:2 ratio of study groups helped to ensure that there were a sufficient number of study participants who would consider attending the CME workshops. The educational intervention Physicians allocated to the intervention arm of MAAUI were mailed a package that included an introduction to the study, audit and feedback material, and an invitation to participate in a CME workshop. Physicians in the control arm received no package. Audit and feedback material The audit and feedback material consisted of a "Personalised Prescribing Profile" (see Figure 2). This profile illustrated for each physician his/her recent prescribing pattern of non-steroidal anti-inflammatory therapies and the appropriateness of this prescribing pattern in light of expert recommendations. Specifically, the profile contained a bar chart showing the number of patients to whom the physician had prescribed long-term treatment with coxibs, traditional NSAID monotherapy, or traditional NSAIDs in combination with GPAs between August 1999 and September 2000. The proportion of these patients with whom the prescription was appropriate was also included. Long-term treatment was defined as patients who received therapy for a minimum of 100 days during this period. The chart, in addition to showing the physician's own prescribing profile, also showed the average profile of primary care physicians in the same geographical area and the profile of Manitoban primary care physicians overall. The reverse side of the chart listed the criteria used to define appropriate prescribing. These criteria were based on international clinical practice guidelines and Manitoba's restricted drug reimbursement policy for coxibs, and were verified by a rheumatologist on the MAAUI research team [18-22]. The criteria identified coxibs or traditional NSAID/GPA combination therapy as the appropriate choice for patients at risk for serious GI complications. The criteria also identified coxibs as the only appropriate therapy for patients with bleeding disorders or patients taking concomitant anticoagulants (this latter recommendation was based in part on Manitoba's reimbursement policy for coxibs and in part on the Canadian Consensus guidelines, and reflected concerns over the anti-platelet effect of traditional NSAIDs [18,22]). The format of the Personalised Prescribing Profiles was developed by the MAAUI research team and was validated prior to the study intervention using two focus groups of family physicians. The Personalized Prescribing Profiles were generated by Manitoba Health and the Manitoba Centre for Health Policy (MCHP) at the University of Manitoba, using administrative data from the Population Health Research Data Repository. This repository contains anonymized encounter-based records of individuals' interactions with the provincial health care system and is derived from information received by the Department of Health, Province of Manitoba, as part of the routine provision of health care in the province. The repository includes Physician Registry files, Medical Claims and Hospital discharge files, as well as Drug Programs Information Network (DPIN) files. The DPIN contains records of all drugs dispensed by Manitoba pharmacies, regardless of who is responsible for payment. Because of the sensitive nature of the Personalised Prescribing Profiles, the individualized profiles were not seen by the MAAUI research team. Instead the packages containing the profiles were assembled and mailed out by Manitoba Health with the assistance of an independent researcher, hired by the MAAUI research team, who signed a confidentiality agreement with Manitoba Health. CME workshop The CME workshop for MAAUI was entitled "The Utilization and Prescribing of Anti-inflammatory Drugs in Osteoarthritis". The workshop focused specifically on the prescribing for osteoarthritis, as it was felt that people with this disease account for a large proportion of the chronic non-steroidal anti-inflammatory use by primary care practices [39]. Eleven CME workshops were held throughout Manitoba (four within Winnipeg and seven outside of Winnipeg) and physicians in the intervention group were invited to voluntarily attend one of the 11. The workshops were free of charge and were approved for 3.0 hours of MAINPRO-M1 credits (continuing education credits awarded by the College of Family Physicians of Canada for group learning activities). Each workshop was facilitated by a trained family physician. The workshops included three components: an introductory video, a decision tree, and a case study portion. The introductory video provided the history and rationale of MAAUI, explained aspects of the Personalised Prescribing Profile, and introduced the decision tree. The decision tree depicted a stepwise approach to the diagnosis and treatment of osteoarthritis. This aid was designed by a rheumatologist on the MAAUI research team specifically for use in the study and was based on international clinical practice guidelines and Manitoba's drug reimbursement policy [18-22]. A take-home copy of the decision tree was provided to each workshop participant (see Additional file 1). Finally, the case study portion of the workshop consisted of studies exploring both the diagnosis and appropriate treatment of osteoarthritis. These case studies were supplied from an existing CME curriculum entitled "Clinical Scenarios in Osteoarthritis" [40]. The overall MAAUI educational intervention, including both the audit and feedback material and the optional CME interactive workshop, was chosen by the MAAUI research team based on the success of similar interventions in the past. Audit and feedback mechanisms have shown moderate success in changing physician behaviour in past studies [41]. Equally, the addition of a continuing education workshop to an existing intervention has been shown to increase the impact on physician behaviour [42]. Workshops with interactive elements, such as case studies, have also been found to be more successful at influencing physician behaviour than purely didactic lectures [43]. Affordability, practicality, and reproducibility were also considered when designing the intervention. Outcomes Principal outcomes The primary goal of MAAUI is to evaluate the impact of the educational intervention on physician appropriate prescribing of non-steroidal anti-inflammatory therapies for patients requiring long-term treatment. To this end, four principal outcome measures have been developed: • the change in appropriate prescribing of all non-steroidal anti-inflammatory therapy (including coxibs, traditional NSAIDs, and traditional NSAID/GPA combination therapy) from pre-to post-intervention; • the change in appropriate prescribing of coxibs from pre-to post-intervention; • the change in appropriate prescribing of traditional NSAID/GPA combination therapy from pre-to post-intervention; and • the change in appropriate prescribing of traditional NSAID monotherapy from pre-to post-intervention. These primary outcomes will be evaluated at the level of the individual physician and will be calculated using administrative data similar to that used to create the Personalised Prescribing Profiles. The change in appropriate prescribing (AP) for a given physician will be calculated by the physician's rate of appropriate prescribing during a 6-month period post-intervention (October 1st 2001 to March 31st 2002) minus this rate during a 10-month period immediately pre-intervention (October 1st, 2000 to July 31st, 2001). The rate of appropriate prescribing will be defined as the number of patients prescribed long-term treatment in whom the treatment was appropriate, divided by the total number of patients prescribed long-term treatment, multiplied by 100%. Otherwise expressed: Physician's ΔAP = Post-intervention AP - Pre-intervention AP Appropriateness of prescribing will be calculated by a researcher who is blinded to participants' study-arm allocation, and will be based on the same criteria as listed in the Personalised Prescribing Profiles (see Figure 2a). In the event that a physician has fewer than five patients on long-term treatment in either the pre- or post-intervention period, that physician's data will be withheld from analysis to ensure confidentiality of the patients. Secondary outcomes In addition to the four principal outcomes, the MAAUI study has a number of prospectively-defined secondary outcomes: • The study will re-evaluate the primary outcomes in the following two physician subgroups: physicians who received the audit and feedback material and attended the CME workshop; physicians who received the audit and feedback material and chose not to attend the CME workshop. This analysis will help to determine the impact that the different components of the educational intervention had on physician behaviour. Specifically in the subgroup of physicians who attended the CME workshop: • The study will measure the change in physician knowledge of osteoarthritis from pre-CME workshop to immediately post-CME workshop and the change in knowledge from pre-CME workshop to five months post-CME workshop. These two outcomes should provide insight into physicians' retention of information following a CME workshop and will allow us to examine any relationship between change in physician knowledge and change in physician prescribing behaviour. • The study will also survey physicians' perceived change in prescribing behaviour at five months post-intervention. This outcome, compared with the principal study outcomes, will provide insight into the accuracy with which physicians' discern their own prescribing practices. • The study will collect process-related measures in order to mark areas for improvement in the study interventions. Specifically, physicians' impressions of the Personalised Prescribing Profile and CME workshop will be surveyed using the questionnaire distributed immediately following the CME workshop and using focus-group discussions at the end of the study follow-up. All workshop participants will be invited to attend a follow-up focus group (one for urban participants and one for rural participants). A separate focus group will be held for the workshop facilitators. Baseline data collection The following data were collected at baseline (October 1st, 2000 to July 31st, 2001) as control measurements: i) physician demographics including sex, urban or rural practice location, and the number of years in practice; ii) physician volume of non-steroidal anti-inflammatory therapies prescribed annually for patients requiring chronic therapy; and iii) physician rate of appropriate prescribing of NSAIDs. These latter two measures were assessed for each of coxibs, traditional NSAID/GPA combination therapy, and traditional NSAID monotherapy. Sample size and statistics Sample size was calculated based on the need to detect at least a 10% improvement in physician practice patterns associated with the intervention, for clinical significance. Alpha error was set at 1% because of the multiple primary outcomes and power was set at 80%. It was also assumed in the sample size calculation that physician baseline demographic measures and baseline practice patterns would contribute an additional 10% variation to the outcomes. Based on these figures and using sample size techniques for multivariate regression analysis [44], we estimated the need for a minimum of 116 physicians in the control group and intervention group respectively. Multivariate regression analysis will be used to determine if the study intervention has a significant impact on the primary outcomes. Baseline data will be included as control measures and interaction effects between these baseline data and the intervention will also be evaluated. Ethics The MAAUI protocol has been approved by the Health Research Ethics Board, Faculty of Medicine, University of Manitoba, and by the Health Information Privacy Committee of Manitoba Health which is responsible for approving research projects that use personal health information held by a government department. Competing interests The MAAUI study is funded by Merck Frosst Canada Ltd., a pharmaceutical company that produces a range of non-steroidal anti-inflammatory therapies including a coxib and various traditional NSAIDs. Merck Frosst's involvement in the study is limited to representation (by L-JM) on the steering committee, which is the governing body responsible for approving all aspects of study development and implementation. Merck Frosst will not participate in data analysis and will not have access to confidential individual patient or physician data. AK, CM, and GT have received speaking fees from Merck Frosst Canada. KR is the scientific writer for this manuscript, employed by the PHCRU. All of these competing interests were not specifically disclosed to study participants. Merck Frosst's sponsorship of the study, however, was disclosed in the consent form signed by participants at the MAAUI CME workshops. Authors' contributions MD, AK, BK, and CM conceived of the study. MD, AK, L-JM, CM, and GT contributed to the development of the protocol. GT created the decision tree and list of criteria for appropriate prescribing. GT also chaired the MAAUI project steering committee. LM is project managing the study. KR performed the literature review for this manuscript and wrote the first draft and coordinated all subsequent revisions. All authors approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Decision Tree: take-home copyThe decision tree was a component of the MAAUI CME workshop. Physicians were introduced to the decision tree during the workshop and were provided with a two-sided take-home copy. The decision tree depicts a stepwise approach to the diagnosis and treatment of osteoarthritis. a/ Side 1. b/ Side 2. Of note, on side 2 of the decision tree the term "NSAID" denotes traditional NSAIDs. click here for file Acknowledgements We are indebted to Health Information Management, Manitoba Health for providing data and to the Manitoba Centre for Health Policy for their maintenance and development of the database. We would also like to acknowledge Manitoba Health for their role on the MAAUI steering committee. The results and conclusions in this paper, however, are those of the authors and no official endorsement by Manitoba Health was intended or should be implied. Figures and Tables Figure 1 MAAUI study design Figure 2 Generic example of a Personalised Prescribing Profile. The Personalised Prescribing Profile constituted the audit and feedback mechanism in MAAUI. Every physician in the intervention arm of the study was sent a Personalised Prescribing Profile by mail. a/ Side 1 of the profile provides a brief introduction to the purpose of the profile and lists the criteria for appropriate prescribing of coxibs, traditional NSAIDs (referred to as "NSAIDs" throughout the Profile), and traditional NSAID/GPA combination therapy. b/ On side 2, a bar chart illustrates for the physician his/her recent prescribing pattern of chronic non-steroidal anti-inflammatory therapies. The chart includes the number of patients to whom the physician has prescribed long-term treatment with each of the anti-inflammatory therapies between August 1999 and September 2000 and the proportion of these patients for whom the prescription was appropriate ("Recommended Treatment") or inappropriate ("Not a recommended treatment"). The chart also provides averaged statistics on appropriate and inappropriate prescribing for the physicians in the same geographical area and for primary care physicians in Manitoba overall. ==== Refs Wolfe MM Lichtenstein DR Singh G Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs N Engl J Med 1999 340 1888 1899 10369853 10.1056/NEJM199906173402407 Laine L Gastrointestinal effects of NSAIDs and coxibs J Pain Symptom Manage 2003 25 S32 S40 12604155 10.1016/S0885-3924(02)00629-2 Bombardier C Laine L Reicin A Shapiro D Burgos-Vargas R Davis B Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. VIGOR Study Group N Engl J Med 2000 343 1520 8,2 11087881 10.1056/NEJM200011233432103 Silverstein FE Faich G Goldstein JL Simon LS Pincus T Whelton A Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study JAMA 2000 284 1247 1255 10979111 10.1001/jama.284.10.1247 Silverstein FE Graham DY Senior JR Davies HW Struthers BJ Bittman RM Misoprostol reduces serious gastrointestinal complications in patients with rheumatoid arthritis receiving nonsteroidal anti-inflammatory drugs. A randomized, double-blind, placebo-controlled trial Ann Intern Med 1995 123 241 249 7611589 Singh G Recent considerations in nonsteroidal anti-inflammatory drug gastropathy Am J Med 1998 105 31S 38S 9715832 10.1016/S0002-9343(98)00072-2 Rahme E Joseph L Kong SX Watson DJ LeLorier J Cost of prescribed NSAID-related gastrointestinal adverse events in elderly patients Br J Clin Pharmacol 2001 52 185 192 11488776 10.1046/j.1365-2125.2001.00348.x Rahme E Joseph L Kong SX Watson DJ LeLorier J Gastrointestinal health care resource use and costs associated with nonsteroidal antiinflammatory drugs versus acetaminophen: retrospective cohort study of an elderly population Arthritis Rheum 2000 43 917 924 10765939 10.1002/1529-0131(200004)43:4<917::AID-ANR25>3.0.CO;2-F Bloom BS Direct medical costs of disease and gastrointestinal side effects during treatment for arthritis Am J Med 1988 84 20 24 3348239 10.1016/0002-9343(88)90250-1 Chevat C Pena BM Al MJ Rutten FF Healthcare resource utilisation and costs of treating NSAID-associated gastrointestinal toxicity. A multinational perspective Pharmacoeconomics 2001 19 17 32 11280103 Herings RM Klungel OH An epidemiological approach to assess the economic burden of NSAID-induced gastrointestinal events in The Netherlands Pharmacoeconomics 2001 19 655 665 11456213 Sturkenboom MC Romano F Simon G Correa-Leite ML Villa M Nicolosi A The iatrogenic costs of NSAID therapy: a population study Arthritis Rheum 2002 47 132 140 11954006 10.1002/art.10268 Hawkey CJ Non-steroidal anti-inflammatory drug gastropathy: causes and treatment Scand J Gastroenterol Suppl 1996 220 124 127 8898449 Maetzel A Krahn M Naglie G The cost effectiveness of rofecoxib and celecoxib in patients with osteoarthritis or rheumatoid arthritis Arthritis Rheum 2003 49 283 292 12794781 10.1002/art.11121 Simon LS Smolen JS Abramson SB Appel G Bombardier C Brater DC Controversies in COX-2 selective inhibition J Rheumatol 2002 29 1501 1510 12136912 Mukherjee D Nissen SE Topol EJ Risk of cardiovascular events associated with selective COX-2 inhibitors JAMA 2001 286 954 959 11509060 10.1001/jama.286.8.954 Solomon DH Glynn RJ Levin R Avorn J Nonsteroidal anti-inflammatory drug use and acute myocardial infarction Arch Intern Med 2002 162 1099 1104 12020178 10.1001/archinte.162.10.1099 Tannenbaum H Peloso PM Russell AS Marlow B An evidence-based approach to prescribing NSAIDs in the treatment of osteoarthritis and rheumatoid arthritis: The Second Canadian Consensus Conference Can J Clin Pharmacol 2000 7 4A 16A 11011110 National Institute for Clinical Excellence Guidance on the use of cyclo-oxygenase (Cox) II selective inhibitors, celecoxib, rofecoxib, meloxicam and etodolac for osteoarthritis and rheumatoid arthritis Technology Appraisal Guidance 2001 27 1 14 Recommendations for the medical management of osteoarthritis of the hip and knee: 2000 update. American College of Rheumatology Subcommittee on Osteoarthritis Guidelines Arthritis Rheum 2000 43 1905 1915 11014340 10.1002/1529-0131(200009)43:9<1905::AID-ANR1>3.0.CO;2-P Guidelines for the management of rheumatoid arthritis: 2002 Update Arthritis Rheum 2002 46 328 346 11840435 10.1002/art.10148 Manitoba Health Prescription Drugs Cost Assistance Act Regulation (Parts 1 and 2) 2003 Ministry of Health Ontario Drug Benefit Formulary / Comparative Drug Index Edition no. 38 2003 VHA Pharmacy Benefits Management Strategic Health Care Group and the Medical Advisory Panel Summary of the criteria for non-formulary use of Cyclooxygenase 2 (COX-II) inhibitors in high-risk veteran patients 2001 Hernandez-Diaz S Rodriguez LA Association between nonsteroidal anti-inflammatory drugs and upper gastrointestinal tract bleeding/perforation: an overview of epidemiologic studies published in the 1990s Arch Intern Med 2000 160 2093 2099 10904451 10.1001/archinte.160.14.2093 Gabriel SE Jaakkimainen L Bombardier C Risk for serious gastrointestinal complications related to use of nonsteroidal anti-inflammatory drugs. A meta-analysis Ann Intern Med 1991 115 787 796 1834002 Schwappach DL Koeck CM Selective COX-2 inhibitors: a health economic perspective Wien Med Wochenschr 2003 153 116 122 12705064 Spiegel BM Targownik L Dulai GS Gralnek IM The cost-effectiveness of cyclooxygenase-2 selective inhibitors in the management of chronic arthritis Ann Intern Med 2003 138 795 806 12755551 Maetzel A Ferraz MB Bombardier C The cost-effectiveness of misoprostol in preventing serious gastrointestinal events associated with the use of nonsteroidal antiinflammatory drugs Arthritis Rheum 1998 41 16 25 9433865 10.1002/1529-0131(199801)41:1<16::AID-ART3>3.3.CO;2-W Lomas J Words without action? The production, dissemination, and impact of consensus recommendations Annu Rev Public Health 1991 12 41 65 2049143 10.1146/annurev.pu.12.050191.000353 Oxman AD Thomson MA Davis DA Haynes RB No magic bullets: a systematic review of 102 trials of interventions to improve professional practice CMAJ 1995 153 1423 1431 7585368 Davis DA Taylor-Vaisey A Translating guidelines into practice. A systematic review of theoretic concepts, practical experience and research evidence in the adoption of clinical practice guidelines CMAJ 1997 157 408 416 9275952 Grimshaw JM Shirran L Thomas R Mowatt G Fraser C Bero L Changing provider behavior: an overview of systematic reviews of interventions Med Care 2001 39 II2 45 11583120 10.1097/00005650-200108002-00002 Cutts C LaCaze A Nonsteroidal anti-inflammatory drugs and potential risks in a convenience sample of general practitioners Aust Fam Physician 2002 31 590 592 12154611 Cox E The Drug of Choice Among Boomers – Cox-2 Inhibitors: Here's What You May Not Know 2002 Mamdani M Rochon P Laupacis A Anderson G Initial patterns of use of COX-2 inhibitors by elderly patients in Ontario: findings and implications CMAJ 2002 167 1125 1126 12427703 Marra CA Esdaile JM Sun H Anis AH The cost of COX inhibitors: how selective should we be? J Rheumatol 2000 27 2731 2733 11128654 Landsberg PG Pillans PI Radford JM Evaluation of cyclooxygenase 2 inhibitor use in patients admitted to a large teaching hospital Intern Med J 2003 33 225 228 12752891 10.1046/j.1445-5994.2003.00392.x Cullen DJ Seager JM Holmes S Doherty M Wilson JV Garrud P Pharmacoepidemiology of non-steroidal anti-inflammatory drug use in Nottingham general practices Aliment Pharmacol Ther 2000 14 177 185 10651658 10.1046/j.1365-2036.2000.00700.x Davis P Hughes S Low S Sholter D Homik J Juby A Clinical Scenarios in Osteoarthritis: Interactive Problem-Based Case Studies 2000 Prepared by the Division of Continuing Medical Education, Faculty of Medicine and Dentistry, University of Alberta, in Edmonton, in partnership with Patient Health – CME Merck Frosst Canada & Co. Jamtvedt G Young JM Kristoffersen DT Thomson O'Brien MA Oxman AD Audit and feedback: effects on professional practice and health care outcomes Cochrane Database Syst Rev 2003 CD000259 12917891 Richards D Toop L Graham P Do clinical practice education groups result in sustained change in GP prescribing? Fam Pract 2003 20 199 206 12651796 10.1093/fampra/20.2.199 Thomson O'Brien MA Freemantle N Oxman AD Wolf F Davis DA Herrin J Continuing education meetings and workshops: effects on professional practice and health care outcomes Cochrane Database Syst Rev 2001 CD003030 11406063 Cohen J Statistical power analysis 1988 2 Hillsdale: Lawrence Erlbaum Associates, Inc

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==== Front Biomed Eng OnlineBioMedical Engineering OnLine1475-925XBioMed Central London 1475-925X-3-281533313210.1186/1475-925X-3-28ResearchReal time electrocardiogram QRS detection using combined adaptive threshold Christov Ivaylo I [email protected] Center of Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev str., blok 105, 1113, Sofia, Bulgaria2004 27 8 2004 3 28 28 4 6 2004 27 8 2004 Copyright © 2004 Christov; licensee BioMed Central Ltd.2004Christov; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background QRS and ventricular beat detection is a basic procedure for electrocardiogram (ECG) processing and analysis. Large variety of methods have been proposed and used, featuring high percentages of correct detection. Nevertheless, the problem remains open especially with respect to higher detection accuracy in noisy ECGs Methods A real-time detection method is proposed, based on comparison between absolute values of summed differentiated electrocardiograms of one of more ECG leads and adaptive threshold. The threshold combines three parameters: an adaptive slew-rate value, a second value which rises when high-frequency noise occurs, and a third one intended to avoid missing of low amplitude beats. Two algorithms were developed: Algorithm 1 detects at the current beat and Algorithm 2 has an RR interval analysis component in addition. The algorithms are self-adjusting to the thresholds and weighting constants, regardless of resolution and sampling frequency used. They operate with any number L of ECG leads, self-synchronize to QRS or beat slopes and adapt to beat-to-beat intervals. Results The algorithms were tested by an independent expert, thus excluding possible author's influence, using all 48 full-length ECG records of the MIT-BIH arrhythmia database. The results were: sensitivity Se = 99.69 % and specificity Sp = 99.65 % for Algorithm 1 and Se = 99.74 % and Sp = 99.65 % for Algorithm 2. Conclusion The statistical indices are higher than, or comparable to those, cited in the scientific literature. ==== Body Background The QRS complexes and ventricular beats in an electrocardiogram represent the depolarization phenomenon of the ventricles and yield useful information about their behavior. Beat detection is a procedure preceding any kind of ECG processing and analysis. For morphological analysis this is the reference for detection of other ECG waves and parameter measurements. Rhythm analysis requires classification of QRS and other ventricular beat complexes as normal and abnormal. Real-time ventricular beat detection is essential for monitoring of patients in critical heart condition. Correct beats recognition is impeded by power-line interference, electromyogram noise and baseline wander often present in the ECG signal. In long-term monitoring electrode impedance can increase considerably, resulting in very low signal-to-noise ratio, which can make detection practically impossible in a single lead. Therefore, usually two or three leads are used for monitoring [1]. Friesen et al. [2] have presented a comparison of nine QRS detection algorithms, based on: i) amplitude and first derivative, ii) first derivative only, iii) first and second derivative, and iv) digital filtering. Daskalov et al. [3] applied these algorithms to selected signals containing records with pronounced baseline drift. The results were unsatisfactory, which was probably due to the use of fixed detection thresholds, whereas adaptive ones would be more appropriate. Poli et al. [4] used a generic algorithm for QRS detection. The complexes were emphasized with respect to the rest of the signal by polynomial filters and compared to an adaptive threshold. The authors reported 99.60 % sensitivity (Se) and 99.51 % specificity (Sp) with the MIT-BIH Arrhythmia Database. The method is inapplicable in real-time. Afonso et al. [5] proposed hardware filter banks for ECG signal decomposition, where several parameters were independently computed and combined in a decision rule. The authors reported Se = 99.59 % and Sp = 99.56 % for their real-time, single-channel beat detection algorithm tested with the MIT-BIH Arrhythmia Database. Dotsinsky and Stoyanov [6] developed a heuristic, pseudo-real-time algorithm for ventricular beat detection for single-channel ECG, based on steep edges and sharp peaks evaluation criteria. They reported Se = 99.04% and Sp = 99.62%, obtained with two channel recordings from AHA and MIT-BIH Arrhythmia Database Moraes et al. [1] combined logically two different algorithms working in parallel – the first has been taken from the work of Englese and Zeelenberg [7] and the other was based on Pan and Tompkins [8], and Ligtenberg and Kunt [9]. Moraes et al. [1] reported Se = 99.22 % and Sp = 99.73 % after having excluded records of patients with pacemaker. After excluding a few more recordings 108, 200, 201 and 203, containing high amplitude noise (according to the authors), the statistical indices rises to Se = 99.56 % and Sp = 99.82 %. Li et al. [10] have used wavelet transforms for detection. They reported 0.15 % false detections out of 46 files from the MIT-BIH Arrhythmia Database, but with exclusion of files 214 and 215. In addition, we found some errors in their Table II. After correction, the reported accuracy slightly decreased. The large variety of QRS detection algorithms, and the continuous efforts for their enhancement, proves that universally acceptable solution has not been found yet. Difficulties arise mainly from the huge diversity of the QRS complex waveforms and the noise and artifacts accompanying the ECG signals. ECG databases All 48 ECG recordings of MIT-BIH Arrhythmia database were used, without exception. Each one has a duration of 30 min and includes two leads – the modified limb lead II and one of the modified leads V1, V2, V4 or V5 [11]. The sampling frequency is 360 Hz with resolution 5 μV/bit. Two cardiologists have annotated all beats. Approximately 70 % of the beats are annotated as Normal. Four of the records are from patients with pacemakers. The American Heart Association (AHA) database was also considered, during the evaluation of the method, mostly due to the fact that it contains patients with premature ventricular beats of contraction of R-over-T type, some of them very difficult to detect because of their closeness to the previous complex. Statistical indices for this database are not derived, because they can be compared with limited number of articles working with AHA. The database consists of 80 recordings: 2-leads, 250 Hz sampling rate and 5 μV/bit resolution. Method The differentiated and summed signals from L leads are compared to the absolute value of a threshold MFR = M + F + R – a combination of three independent adaptive thresholds, where: • M – Steep-slope threshold; • F – Integrating threshold for high-frequency signal components; • R – Beat expectation threshold. Two algorithms were developed: Algorithm 1 detects at the current beat. Algorithm 2 Pseudo-real-time detection with additional triggering of potentially missed heart beat in the last interval by RR interval analyses. The algorithms are self-adjusting to the thresholds and weighting constants, regardless of resolution and sampling frequency used. They operate with any number L of ECG leads, self-synchronize to QRS or beat slopes and adapt to beat-to-beat intervals. Preprocessing • Moving averaging filter for power-line interference suppression: averages samples in one period of the power-line interference frequency with a first zero at this frequency. • Moving averaging of samples in 28 ms interval for electromyogram noise suppression – a filter with first zero at about 35 Hz. • Moving averaging of a complex lead (the sintesis is explained in the next section) in 40 ms intervals – a filter with first zero at about 25 Hz. It is suppressing the noise magnified by the differentiation procedure used in the process of the complex lead sintesis. Complex lead The algorithm operates with a complex lead Y of several primary leads L. In cases of 12-standard leads, synthesis of the three quasi-orthogonal Frank leads is recommended first [3,12], thus determining the complex lead as a spatial vector. The complex lead is obtained as: where Xj(i) is the amplitude value of the sample i in lead j, and Y(i) is the current complex lead. The above formula (except the normalizing coefficient 1/L and the absolute value) was initially adopted from the work of Bakardjian [13]. Operating with unsigned (absolute) values proved convenient when dealing with QRSs and extrasystoles having different, for example positive (in one lead) and negative (in the other lead) deflections. Adaptive steep-slope threshold – M • Initially M = 0.6*max(Y) is set for the first 5 s of the signal, where at least 2 QRS complexes should occur. A buffer with 5 steep-slope threshold values is preset: MM = [M1M2M3M4M5], where M1 ÷ M5 are equal to M. • QRS or beat complex is detected if Yi ≥ MFR, • No detection is allowed 200 ms after the current one. In the interval QRS ÷ QRS+200ms a new value of M5 is calculated: newM5 = 0.6*max(Yi) The estimated newM5 value can become quite high, if steep slope premature ventricular contraction or artifact appeared, and for that reason it is limited to newM5 = 1.1* M5 if newM5 > 1.5* M5. The MM buffer is refreshed excluding the oldest component, and including M5 = newM5. M is calculated as an average value of MM. • M is decreased in an interval 200 to 1200 ms following the last QRS detection at a low slope, reaching 60 % of its refreshed value at 1200 ms. • After 1200 ms M remains unchanged. The thresholds definitions are presented in more detail with the help of several examples. Two ECG leads are shown in Fig. 1a. Detected QRSs are marked with 'red O' on Lead 1. The summary lead and the steep-slope threshold are represented in Fig. 1b. Figure 1 Adaptive steep-slope threshold Adaptive integrating threshold – F The integrating threshold F is intended to raise the combined threshold if electromyogram noise is accompanying the ECG, thus protecting the algorithm against 'erroneous beat detection'. Initially F is the mean value of the pseudo-spatial velocity Y for 350 ms. With every signal sample, F is updated adding the maximum of Y in the latest 50 ms of the 350 ms interval and subtracting maxY in the earliest 50 ms of the interval. F = F + (max(Yin latest 50 ms in the 350 ms interval) - max(Yin earliest 50 ms in the 350 ms interval))/150 The way F is updated means that not every sample in the interval is integrated, but just the envelope of the pseudo-spatial velocity Y. The weight coefficient 1/150 is empirically derived. Two ECG leads are shown in Fig. 2a. The pseudo-spatial velocity Y and the integrated threshold are presented in Fig. 2b. The correct detection is due to the rise of F (hence of MFR) with about 0.2 mV. The beat complex is included in the integration process (note the high rise of F after any of the complexes), thus making almost impossible a close detection to the previous complex. Figure 2 Adaptive integrating threshold Adaptive beat expectation threshold – R The beat expectation threshold R is intended to deal with heartbeats of normal amplitude followed by a beat with very small amplitude (and respectively with very small slew rate). This can be observed for example in cases of electrode artifacts. Conversely to the integrating threshold protecting against erroneous QRS detection, R is protecting against 'QRS misdetection'. A buffer with the 5 last RR intervals is updated at any new QRS detection. Rm is the mean value of the buffer. • R = 0 V in the interval from the last detected QRS to 2/3 of the expected Rm. • In the interval QRS + Rm * 2/3 to QRS + Rm, R decreases 1.4 times slower then the decrease of the previously discussed steep slope threshold (M in the 200–1200 ms interval). • After QRS + Rm the decrease of R is stopped. The time-course of the beat expectation threshold R is shown in Fig. 3. The decrease of R (respectively MFR) with about 0.2 mV at the fourth QRS allows its detection, despite the lack of complex in Lead 2, which leads to a two-fold decrease of the summary lead amplitude Y (Fig. 3b). Figure 3 Adaptive beat expectation threshold Combined adaptive threshold – MFR The combined adaptive threshold is a sum of the adaptive steep-slope threshold, adaptive integrating threshold and adaptive beat expectation thresholds. (Fig. 4) Figure 4 Combined adaptive threshold MFR = M + F + R Algorithm 2: pseudo-real-time detection with additional triggering of eventually missed heart beat in the last detected RR interval All previous considerations relate to Algorithm 1, which detects a beat at its occurrence. Additional checking for an eventually missed heartbeat is performed by Algorithm 2. Its function is explained by the signal in Fig. 5. The fourth complex at the 15.2 s in Fig. 5b should be missed due to the fact that, MFR is greater then the summary lead Y. Figure 5 Pseudo-real-time detection with additional triggering of eventually missed heart beat in the last RR interval. Let's mark the previous RR interval with t1 and the last – with t2 (Fig. 5a). If t1 is not shortened, which is tested by logic OR of the 2 conditions t1>Rm OR Rm-t1<0.12*Rm AND in the same time t2 is quite long to fulfill the condition abs(t2-2*Rm)<0.5*Rm, the interval is subjected to check for a missed complex. A test is performed on each of the primary leads where a sharp peak is searched (defined as a product > 4 μV of two signal differences having one central and two lateral points 8 ms apart). If the test is passed, a second one is carried out for the amplitude of the summary lead at that point, which should be bigger then 1/3 of the mean value of the buffer MM, in order to define this point as a missed QRS complex. Results and discussion Normally the statistical indices Se and Sp are derived from the following parameters: correctly detected beats TP (true positive), falsely detected beats FP (false positive) and undetected beats FN (false negative). In addition, we used two parameters, adopted from Dotsinsky and Stoyanov [6], as described below. SP – shifted positive error was introduced in order to explain cases like the one shown in Fig. 6. Here the algorithm made a false positive error before the 3rd QRS and missed the following QRS. Formally, this is a false positive error, immediately followed by a false negative. However, if the time interval between these two detections is reasonably short, for example ≤ 200 ms, we accepted this as one error only, labeled as Shifted False Positive Error (SP). Figure 6 Shifted positive error at the P wave Another example of SP error as a result of artifacts just before the normal complexes is shown in Fig. 7. Figure 7 Shifted positive errors, false positive + false negative twins SN – shifted negative error was assumed by the same principle as SP, but in the opposite sense. It also included twin FN+FP error occurring within 200 ms. The first incoming FP or FN error of the shifted is defining it as SP or SN. The logic of using shifted errors (instead of FP and FN or FN and FP in cases when they appear within 200 ms of each other) is that thus the total number of beats in a record retains its value. Otherwise it would change depending on the type and number of errors and thus impede correct computation of Se and Sp. The sensitivity Se is calculated by summing FN SN, while the specificity Sp – by summing FP+SP. The method was developed in Matlab. All 48 recordings from the MIT-BIH Arrhythmia database, without any exception, were used for testing the two algorithms. The processed files containing detection marks were automatically compared with the original MIT-BIH annotated beats by specially designed software. It shows all cases where the annotation and detection marks differ by more than 60 ms. These cases were examined by an independent expert, thus excluding possible author's influence. The results for the two algorithms are presented in Table 1. Table 1 Statistical results for the two algorithms File Annotated beats Algorithm 1 Algorithm 2 TP FN FP SN SP TP FN FP SN SP 100 2273 2273 0 0 0 0 2273 0 0 0 0 101 1863 1862 1 4 0 0 1862 1 4 0 0 102 2187 2187 0 0 0 0 2187 0 0 0 0 103 2084 2062 2 54 11 9 2065 0 58 12 7 104 2212 2211 1 0 0 0 2211 1 0 0 0 105 2567 2543 2 35 8 14 2544 2 36 8 13 106 2027 2017 1 1 0 9 2018 0 1 0 9 107 2137 2135 2 0 0 0 2137 0 0 0 0 108 1763 1664 2 40 3 94 1674 1 42 3 85 109 2532 2521 11 1 0 0 2527 5 0 0 0 111 2124 2124 0 0 0 0 2124 0 0 0 0 112 2539 2539 0 0 0 0 2539 0 0 0 0 113 1797 1797 0 0 0 0 1797 0 0 0 0 114 1879 1879 0 0 0 0 1879 0 0 0 0 115 1953 1951 0 4 1 1 1952 0 4 0 1 116 2412 2389 22 2 0 1 2392 19 2 0 1 117 1535 1535 0 0 0 0 1535 0 0 0 0 118 2275 2275 0 0 0 0 2275 0 0 0 0 119 1987 1987 0 0 0 0 1987 0 0 0 0 121 1863 1863 0 0 0 0 1863 0 0 0 0 122 2476 2476 0 0 0 0 2476 0 0 0 0 123 1518 1516 2 0 0 0 1516 2 0 0 0 124 1619 1617 2 0 0 0 1619 0 0 0 0 200 2601 2549 9 39 18 25 2552 6 41 20 23 201 1963 1902 60 0 0 1 1902 60 0 0 1 202 2136 2130 6 0 0 0 2130 6 0 0 0 203 2978 2901 71 13 3 3 2911 62 27 3 2 205 2656 2652 4 0 0 0 2652 4 0 0 0 207 1862 1860 2 0 0 0 1862 0 1 0 0 208 2954 2937 14 7 2 1 2939 11 7 2 2 209 3004 3004 0 1 0 0 3004 0 1 0 0 210 2647 2591 56 1 0 0 2603 44 1 0 0 212 2748 2748 0 0 0 0 2748 0 0 0 0 213 3551 3548 3 0 0 0 3550 1 0 0 0 214 2260 2258 1 1 1 0 2256 4 1 0 0 215 3362 3362 0 0 0 0 3362 0 0 0 0 217 2208 2204 3 0 0 1 2205 2 0 0 1 219 2154 2153 1 0 0 0 2153 1 0 0 0 220 2048 2048 0 0 0 0 2048 0 0 0 0 221 2427 2426 1 0 0 0 2426 1 0 0 0 222 2483 2480 2 0 0 1 2482 0 0 0 1 223 2595 2585 10 0 0 0 2590 5 0 0 0 228 2053 2053 0 0 0 0 2053 0 1 0 0 230 2256 2256 0 0 0 0 2256 0 0 0 0 231 1886 1886 0 0 0 0 1886 0 0 0 0 232 1767 1766 0 12 0 1 1766 0 12 0 1 233 3076 3073 3 0 0 0 3074 2 0 0 0 234 2753 2753 0 0 0 0 2753 0 0 0 0 Sum 110050 109548 294 215 47 161 109615 240 239 48 147 Of all 110050 annotated beats ('unknown' or 'questionable' were excluded from the study), true detected are 109548 for Algorithm 1 and 109616 for Algorithm 2. The statistical indices are: Algorithm 1: Se = 99.69 %, Sp = 99.66 %; Algorithm 2: Se = 99.74 %, Sp = 99.65 %. The standard way of Se and Sp calculation, considering the joint SP and SN errors as two separate errors gives the following results: Algorithm 1: Se = 99.54 %, Sp = 99.61 %; Algorithm 2: Se = 99.60 %, Sp = 99.60 %. Algorithm 2 improved the sensitivity by 0.05 % (0.06 % for the standard evaluation) as a result of decreased number of undetected beats. This result can be observed for example in recordings 109,203, 210 and 223, where the additionally detected beats are respectively 6, 9, 12 and 5. The performance of both algorithms was especially tested with the file A5001 from the AHA containing R-over-T premature ventricular complexes, very close to the previous normal QRS complex (Fig. 5a). An improvement of 74 undetected by Algorithm 1 R-on-T complexes was observed. The detection of such premature ventricular complexes occurring at the time of ventricular repolarization was considered important, having in mind possible risk of ventricular fibrillation triggering by R-on-T events. Conclusions The proposed algorithms for real-time and pseudo-real-time implementation are adaptive, independent of thresholds and constants values. They are self-synchronized to the QRS steep slope and the heart rhythm, regardless of the resolution and sampling frequency used. Due to the integration threshold, the algorithms are practically insensitive to electromyogram and similar high-frequency noise. The algorithms can operate with one, two or more leads, using a combined lead signal derived from the sum of absolute values of the differentiated lead signals. The statistical indices are higher than, or comparable to those, cited in the scientific literature. Acknowledgements The author gratefully acknowledges the contribution of Prof. Ivan Dotsinsky, Centre of Biomedical Engineering, Bulgarian Academy of Sciences, for examining and testing the results of the proposed algorithms. ==== Refs Moreas JCTB Seixas MO Vilani FN Costa EV A QRS complex detection algorithm using electrocardiogram leads Comp in Card 2002 29 205 208 Friesen GM Jannett TC Jadallah MA Yates SL Quint SR Nagle HT A comparison of the noise sensitivity of nine QRS detection algorithms IEEE Trans on Biomed Eng 1990 37 85 98 10.1109/10.43620 Daskalov IK Dotsinsky IA Christov II Developments in ECG acquisition, preprocessing, parameter measurement and recording IEEE Eng in Med & Biol 1998 17 50 58 10.1109/51.664031 Poli R Cagnoni S Valli G Generic design of optimum linear and nonlinear QRS detection IEEE Trans on Biomed Eng 1995 42 1137 1141 10.1109/10.469381 Afonso VX Tompkins WJ Nguyen TQ Luo S ECG beat detection using filter banks Trans Biomed Eng 1999 2 192 201 10.1109/10.740882 Dotsinsky I Stoyanov T Ventricular beat detection in single channel electrocardiograms BioMed Eng OnLine 2004 3 3 14750981 10.1186/1475-925X-3-3 Englese WAH Zeelenberg C A single scan algorithm for QRS detection and feature extraction IEEE Comp in Card 1979 37 42 Pan J Tompkins W A real-time QRS detection algorithm IEEE Trans Biomed Eng 1985 32 230 236 3997178 Ligtenberg A Kunt M A robust-digital QRS detection algorithm for arrhythmia monitoring Computers and Biomed Res 1983 16 273 286 10.1016/0010-4809(83)90027-7 Li C Zheng C Tai C Detection of ECG characteristic points using wavelet transforms IEEE Trans on Biomed Eng 1995 42 21 28 10.1109/10.362922 Mark R Moody G MIT-BIH Arrhythmia data base directory Cambridge: Massachusetts Institute of Technology 1988 Levkov C Orthogonal electrocardiogram derived from the limb and chest electrodes of the conventional 12-lead system Med Biol Eng Comput 1987 25 155 164 3695618 Bakardjian H Ventricular beat classifier using fractal number clustering Med Biol Eng Comput 1992 30 495 502 1284086

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==== Front Int J Health GeogrInternational Journal of Health Geographics1476-072XBioMed Central London 1476-072X-3-181533313110.1186/1476-072X-3-18MethodologyExploratory disease mapping: kriging the spatial risk function from regional count data Berke Olaf [email protected] Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario, CANADA, N1G 2W12 Department of Biometry, Epidemiology and Information Processing, School of Veterinary Medicine Hannover, Bünteweg 2, D-30559 Hannover, Germany2004 26 8 2004 3 18 18 22 7 2004 26 8 2004 Copyright © 2004 Berke; licensee BioMed Central Ltd.2004Berke; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background There is considerable interest in the literature on disease mapping to interpolate estimates of disease occurrence or risk of disease from a regional database onto a continuous surface. In addition to many interpolation techniques available the geostatistical method of kriging has been used but also criticised. Results To circumvent these critics one may use kriging along with already smoothed regional estimates, where smoothing is based on empirical Bayes estimates, also known as shrinkage estimates. The empirical Bayes step has the advantage of shrinking the unstable and often extreme estimates to the global or local mean, and also has a stabilising effect on variance by borrowing strength, as well. Negative interpolates are prevented by choice of the appropriate kriging method. The proposed mapping method is applied to the North Carolina SIDS data example as well as to an example data set from veterinary epidemiology. The SIDS data are modelled without spatial trend. And spatial interpolation is based on ordinary kriging. The second example is included to demonstrate the method when the phenomenon under study exhibits a spatial trend and interpolation is based on universal kriging. Conclusion Interpolation of the regional estimates overcomes the areal bias problem and the resulting isopleth maps are easier to read than choropleth maps. The empirical Bayesian estimate for smoothing is related to internal standardization in epidemiology. Therefore, the proposed concept is easily communicable to map users. ==== Body Background As with the analysis of any set of data, it is always good practice to begin by producing and inspecting graphs. A feel for the data can then be obtained and any outstanding features identified. In spatial epidemiology this is called disease mapping. Bithel [7], Diggle [14] and Lawson [25] provide recent reviews of disease mapping. Spatial epidemiology comprises at least three types of study focus [17,25]. These are (i) disease mapping, (ii) disease clustering and (iii) geographical correlation analysis but these distinctions are not strict. For example a disease map is also used for reporting the results of a geographical correlation study or to highlight areas of high or low disease incidence, i.e. cluster locations in a cluster study [4,14]. But in the following, disease mapping is considered as exploratory analysis used to get an impression of the geographical or spatial distribution of disease or the corresponding risk. For this the disease map should be based on smoothed estimates, clean of noise and adjusted for variation in 'at-risk' population [[24], p. 163]. The resulting disease map should provide insight into possible causes, effects and trends in the vast amount of data. This will provide an invaluable starting point for epidemiologic enquiry. There are three basic types of disease maps corresponding to certain types of data. These are dot maps for point (or case-event) data, choropleth maps for regional data (also called lattice- or census-tract data), and lastly, isopleth maps for geostatistical data (also called point measurements) representing spatially continuous phenomena at a limited number of sampling locations. Spatial epidemiology is mainly concerned with the analysis of two types of data: case-event data and regional summary data, generally leading to dot maps and choropleth maps, respectively. However, epidemiology is also concerned with the identification of unknown risk factors, which may be part of the environment such as air pollution, radiation or magnetic fields. And such factors vary spatially continuously. Thus it is important to produce an isopleth map of disease occurrence or risk. Furthermore, as has been often pointed out in the literature [[24], p. 131], choropleth maps must be interpreted with caution, as the grey scale grouping is arbitrary and such a choice can affect interpretation, although choropleth maps imply a constant risk (incidence or prevalence) over regions with discontinuities at the border lines. Another point of criticism concerns the areal bias [12], as the varying shape and size of geographical regions make the patchy maps difficult to interpret. The visual impact of larger areas is higher and may dominate the map, leading to biased visual perception, whereas in human epidemiology it is the smaller, urban areas, and not the rural surroundings, that are of primarily interest due to population sizes. These objections may be circumvented by the use of continuous surface mappings, i.e. isopleth maps. Methods Review of interpolation methods For spatial point case-control data Bithell [6] introduced a spatial interpolation method based on kernel density estimation. The resulting map is called the "spatial relative risk function". Lawson and Williams [28] and Kellsall and Diggle [20] proposed modifications of this procedure. Regional data arise from summarising individual information for administrative regions such as census tracts. The basic model for the individual data, i.e. spatial point data, is the spatial Poisson process. In case of a rare disease, aggregation of the data over distinct regions results again in Poisson distributed data. For a more common disease the regional counts may be binomially distributed. When spatial interpolation of regional data is the objective of the disease mapping study a grid of surface interpolant co-ordinates must be provided. Then a number of techniques can be used to automatically interpolate the data by use of deterministic methods or to predict the values statistically at the grid co-ordinates. Among the possible choices are kernel smoothing, splines, loess and running medians; see [19] for a discussion of these methods. Kernel smoothing has the advantage of preserving the positivity condition implied in rate data. Brillinger [8] used kernel-type smoothing in the context of birth rate data, and Müller, Stadtmüller and Tabnak [33] applied locally weighted least squares adapted for spatial aggregation to AIDS incidence maps for the San Francisco Area. Another approach for the interpolation of regional data onto a continuous surface is the geostatistical prediction method of kriging. Some implementations of kriging have been proposed to obtain a risk surface [10,23,31,34,35,38]. Kelsall and Wakefield [21] have proposed a more complex geostatistical approach based on generalised linear modelling (GLIM) of regional data, which is similar to the work by Diggle, Tawn and Moyeed [15]. The relative merits of different interpolation approaches haves not so far been systematically investigated [[24], p. 131]. Kriging the spatial risk function The problems with the kriging method for generating isopleth maps of disease occurrence, i.e. the spatial risk function, are: (1) heterogeneous variances in the regional estimates and (2) the potential of negative interpolations. The first problem can be ameliorated by the use of empirical Bayes estimation to smooth the data prior to kriging. Appropriate geostatistical modelling can solve the second problem. Let the study area be divided into N disjunctive sub regions indexed by i (i = 1,..., N). Define ni as the size of the 'at-risk' population of the i-th region, and denote the number of cases by mi. The proportion pi = mi/ ni is the crude estimate for the parameter of interest θi. Smoothing by empirical Bayes estimation Mapping raw estimates of disease occurrence can lead to spurious spatial features. To overcome this problem Cressie and Read [13] have explored the use of several variance stabilising transformations, but the results on the transformed scale are difficult to interpret. Furthermore, empirical Bayesian methods have been developed based on the idea of pooling information across regions. The resulting smoothed regional estimates have a variance stabilising side effect by borrowing strength from (local or global) neighbourhood information. The outline of the empirical Bayes approach for smoothing regional rates of rare diseases is based on the Poisson model. For the case of more common diseases Martuzzi and Elliott [30] adapted the approach to the Binomial model. Poisson model for rare diseases Clayton and Kaldor [11] proposed empirical Bayes or shrinkage estimation for smoothing regional data along with maximum likelihood estimation (MLE) of the unknown prior parameters. Marshall [29] modified this approach using method of moments estimators (MME) instead of MLE. The resulting estimates provide starting values for the iterative maximum likelihood procedure; or they could be used for exploratory mapping purposes, which is the main interest of this work. Assume the cases in every region i are independently Poisson distributed with the unknown parameter θi which has an unknown prior distribution associated with expectation E(θi) = π and variance Var(θi) = φ2. Then the totals of cases from the i-th region, e.g. mi (i = 1,..., N), are distributed as follows mi | θi, ni ~ Po(ni θi) θi ~ (π, φ2) The MME of the unknown hyperparameters are for the prior mean and for the prior variance, where denotes the mean regional 'at-risk' population and the summation is over the range of i. The empirical Bayesian estimates then becomes with shrinkage weights . Tendency of variance homogeneity As stated above, the smoothing of the crude regional estimates pi has the side-effect of stabilising the variances for the regional empirical Bayes estimates, i.e. . Heuristically this is clear, because the empirical Bayes estimates are based on the whole sample information and not just on the individual regional sample, and thus are more stable. But this is difficult to prove, because this requires an analytical expression for the corresponding variance or an approximate variance estimate. Morris [32] proposed an approximate variance estimate for the empirical Bayes estimate in the Gaussian/Gaussian setting. However, an extension of this idea to the non-Gaussian setting is awkward [[9], p. 80]. Therefore, two facts are used to claim the conjecture that the empirical Bayes estimates of the regional estimates show up the tendency of variance homogeneity. First, it should be noted that Bayes estimates, i.e. , generally have a smaller associated variance than the corresponding frequentist estimates, i.e. pi [32]. This means the Bayesian smoothing generally results in reduced variances, thus reducing the absolute differences between regional variances, i.e. the variance heterogeneity. Secondly, by virtue of the shrinkage property of the empirical Bayes estimate, it follows that the variance will be shrunk back to the global variance in the case of small regional samples, which is responsible for a large part of the unstable estimates. Both points together make up what is called "borrowing strength from the ensemble" [32]. Geostatistical modelling The geostatistical method of kriging is widely accepted for the purpose of spatial prediction, i.e. interpolation and (moderate) extrapolation. It is proposed here to predict the smoothed regional data onto a fine meshed regular grid of points for isopleth mapping purposes. Spatial linear model Kriging of spatial data, say Z = (Z1,..., ZN)' at sample sites si, i = 1,..,N, takes place within the framework of the spatial linear model [1,12] Z = μ + δ,     E(Z) = μ,     δ ~ Gau(0, Σ) An integral part of this model is spatial correlation, which must be taken into account to draw valid scientific inferences. Here Σ is a variance-covariance matrix, spatially structured according to the position and direction between sampling sites. The basic assumption for spatial data is that near things are more related than distant things. This was neglected for smoothing of the regional epidemiological measures (prevalence or incidence) via empirical Bayes estimators. Semivariogram For geostatistical modelling, the structure of spatial variation will be estimated through the semivariogram , where h denotes the translation between any two arbitrary sites si and sj within the study region. See [12,37] for diverse parameterised semivariogram models and estimation methods. Kriging In geostatistics kriging is used synonymously with optimal spatial prediction. However the optimality depends on the appropriateness of the spatial model. Cressie [12] gives a review on diverse kriging approaches. There are different models with respect to the knowledge and estimation of the spatial mean function, i.e. μ(s). Ordinary kriging is concerned with an unknown but constant mean function, i.e. μ(s) = μ. Furthermore, universal kriging is based on a polynomial trend surface model which is to be removed prior to estimation of the semivariogram from the residuals, i.e. δ(s). This technique may be the most widely-used in practice. An outlier-resistant alternative is median polish kriging. This method starts by the robust and non-parametric estimation of the non-constant mean surface via median polishing followed by robust semivariogram estimation. Berke [2] proposed a modification of median polish kriging for larger spatial data sets. Generally, kriging surfaces are the sum of an estimate for the trend surface μ(s) plus the kriging prediction for the residual process δ (s), formally . Kriging and smoothing Kriging is sometimes termed a smoothing method. This is due to the fact that the predicted residuals are in absolute not larger than the model residuals δ(s) = Z(s) - μ(s), i.e. the variability of the predictions around the estimated mean surface is smaller than the variability of the observations Z(s). When the semivariogram is modelled without nugget effect, i.e. without small-scale variability at spatial scales smaller than the observational scale, then kriging leads to direct interpolation at the sampling sites. In this case the prediction equals the observation at the sample sites and thus the predicted residuals are equal to the model residuals, i.e. . Predictions at any other sites have the tendency to shrink towards the value of the estimated trend surface at that place. On the other hand, when a semivariogram with nugget effect is appropriate, than the prediction tends to be closer to the mean surface, which gives smaller residuals , i.e. a smoother prediction surface . Thus the prediction of invalid values for risk or for other epidemiologic measures, as has been criticised in the past, is a consequence of inappropriate spatial modelling. This could only happen when universal kriging is based on an estimated trend surface model , which exceeds the range of valid values. Results The mapping technique proposed to generate isopleth risk maps from regional count data is now applied to two example data sets. Example 1 is based on the SIDS mortality rates in North Carolina that show no spatial trend. Example 2 is based on spatial trend contaminated data of tapeworm infections among red foxes in Lower Saxony. Example 1: sudden infant death syndrome (SIDS) in North Carolina This by now classical spatial data set on SIDS mortality rates in North Carolina from 1974 to 1984 has been analysed by many researchers. Cressie [12] gives an introduction to the problem, earlier references and results on data modelling, mapping and cluster detection. More recently Kulldorff [22] applied the spatial scan test to detect clusters of disease and Lawson and Clark [27] applied a spatial mixture modelling approach to map the standardized mortality ratio (SMR) for the period 1974 – 1978. For the years 1974 to 1984 the number of live births ranges from 567 to 52345 over North Carolina's 100 counties. The total number of reported SIDS cases is 1503 out of 753354 live births, which results in an annual mortality rate of approximately 2 per 1000 live births. The mean of the counties boundary files coordinates in longitude and latitude were used here as the geographic coordinates for the regions centres. Figure 1 shows the shrinkage effect by using parallel box plots for the raw rates and the empirically Bayesian smoothed rates under the Poisson model for rare phenomena. Figure 1 Comparison of raw and Bayesian smoothed SIDS mortality rates. Parallel box plots for the raw annual SIDS mortality rate per 1000 live births (r) and the corresponding shrinkage estimates or empirical Bayesian estimated rates (eber) from 100 counties of North Carolina, 1974–1984. The smoothed rates are more appropriate for disease mapping than the raw rates and hence used here for choropleth mapping in Figure 2. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. These are used to highlight the upper and lower five percent of the distribution of the Bayesian smoothed mortality rates and to distinguish between higher and lower values. The smoothed rates vary from about 1.2 to 3.5 cases per 1000 live births. Visual map perception reveals some potential high and low risk areas in the north and southwest but no striking spatial trend pattern. Figure 2 Choropleth map of Bayesian smoothed SIDS mortality rates of North Carolina. Choropleth map of the empirical Bayesian smoothed mortalities per 1000 live births from 100 counties of North Carolina, 1974–1984. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. Geostatistical prediction, i.e. kriging of the empirical Bayesian smoothed rates, is based on appropriate modelling of the data. Here, the constant mean assumption for the spatial mean surface is chosen and justified by visual inspection of the empirical semivariogram which levels out and reaches a sill. The spatial dependence structure is modelled by an isotropic exponential semivariogram without nugget effect, which is fitted by weighted least squares estimation to the robustly estimated empirical semivariogram; see [12] for technical details. The result of this procedure is summarised in Figure 3. The close fitting of the empirical semivariogram to the model indicates appropriate model choice for the dependence structure as well as for the constant mean surface. See [1] for diagnostic methods and its applications in geostatistical modelling. Figure 3 Empirical semivariogram from smoothed SIDS mortality rates and fitted exponential model. Exponential model (sill = 0.22, range = 0.37) fitted by weighted least squares (WLSE) to the robust empirical semivariogram from empirical Bayesian smoothed annual SIDS mortality rates per 1000 live births from 100 counties of North Carolina, 1974–1984. Due to the constant mean assumption ordinary kriging is the appropriate spatial prediction method and without nugget effect this leads to direct interpolation of the data at the counties centres and to smoothed values shrunken towards the global mean for the rest of the study area. The resulting isopleth map or risk surface map is given in Figure 4. Now the grey scale shading is almost continuous, i.e. the patchy nature of Figure 2 is replaced by a surface. Additional isolines are drawn for the same cut points as for the grey scale shadings in Figure 2 (i.e. at the 5%, 50% and 95% level of the empirical distribution of the empirical Bayesian estimates) and will be useful to support map interpretation and comparison. The risk surface map may be more useful to identify potential environmental risk factors than the choropleth map. Figure 4 Isopleth map from kriging the smoothed SIDS mortality rates of North Carolina. Isopleth map based on kriging predictions of the empirical Bayesian smoothed annual SIDS mortality rates per 1000 live births from 100 counties of North Carolina, 1974–1984. Cross-validation can be used to explore the predictive performance of the kriging model. The cross-validation residuals (weighted with respect to the spatial dependence structure given by the semivariogram) should be approximately Gaussian distributed. Normal probability plots can be used to disclose grossly model inadequacies [[12], p. 498] as well as for outlier identification. Figure 5 shows the normal probability plot of the cross-validation residuals based on the refitted model with the outliers removed. Figure 5 Normal probability plot of cross-validation residuals from kriging smoothed SIDS mortality rates. Normal probability plot of the cross-validation residuals from kriging the empirical Bayesian smoothed regional SIDS mortality rates. Observations 4, 42, 83, 8 and 24 as well as 77 and 59 may be outliers with respect to the spatial model. Aside from some positive and negative extreme values the general appearance of the residual process is Gaussian. The potential outliers are regions with steep gradients in risk and hence part of disease clusters (or their surroundings) which were previously identified [22] and are located in the north and south-west of North Carolina. Of course, disease clusters are of interest and the proposed modelling approach reveals their existence and points to the respective high risk areas. This is in line with the gross aims of exploratory disease mapping. Example 2: tapeworm infections in red foxes in Lower Saxony Echinococcus multilocularis (E.m.) is a tapeworm occurring in the northern hemisphere, including endemic regions in central Europe, most of northern and central Eurasia and parts of North America. In central Europe the red fox is the main definitive host with rodents such as mice or muskrats serving as intermediate hosts [16]. The parasite E.m. causes the zoonosis alveolar echinococcosis (A.E.), which has a potentially high fatality rate. Recent studies reflect an alarmingly wide geographic range of the parasite in foxes, with average prevalences varying up to 60% for central Europe. However, the spatial distribution of E.m. in foxes is complex and insufficiently known. There are indications of emerging risk factors for human A.E. such as increasing parasite prevalences in red foxes, growing fox population and progressive spread of foxes to cities. The federal state of Lower Saxony is part of northern Germany and contains the federal city-state of Bremen as an enclave. During the period from 1991 to 1997, 5365 red foxes were sampled in Lower Saxony and examined for infections with E.m.. The data are given in Table 1[3]. Table 1 Regions and statistics for the fox tape worm example. The 43 investigated regions in Lower Saxony with their co-ordinates (x, y), the number of red foxes tested (n) and found positive for E. multilocularis (m), the raw period prevalence in % (PP) and the empirical Bayes smoothed period prevalence in % (BPP). The regions no. 5, 8, 13, 14 and 15 form a previously identified disease cluster [4]. Regions Statistics Nr. Name x y n m PP BPP 1 Braunschweig 7,57 -4,30 25 1 4 6 2 Salzgitter & Wolfenbüttel 7,47 -6,07 115 9 8 8 3 Wolfsburg 9,33 -2,83 22 6 27 23 4 Gifhorn 8,15 -1,37 158 13 8 8 5 Göttingen 3,44 -12,92 157 84 54 51 6 Goslar 6,68 -8,81 152 20 13 13 7 Helmstedt 10,11 -4,30 66 7 11 10 8 Northeim 2,86 -10,28 186 96 52 49 9 Osterode/Harz 6,68 -11,07 94 23 24 23 10 Peine 5,80 -4,11 115 10 9 9 11 Hannover 2,17 -2,64 327 41 13 12 12 Diepholz -4,39 0,88 143 10 7 7 13 Hameln-Pyrmont -0,17 -6,36 99 41 41 39 14 Hildesheim 3,74 -6,36 202 60 30 29 15 Holzminden 1,19 -8,62 60 16 27 24 16 Nienburg/Weser -1,74 -0,58 325 20 6 6 17 Schaumburg -1,25 -4,11 90 12 13 13 18 Celle 4,81 0,59 255 8 3 3 19 Cuxhaven -3,51 10,69 73 14 19 18 20 Harburg 3,64 7,06 285 17 6 6 21 Lüchow-Dannenberg 11,68 3,92 225 20 9 9 22 Lüneburg 7,37 6,37 278 22 8 8 23 Qsterholz-Scharmbeck -3,80 6,57 32 8 25 22 24 Rothenburg/Wümme -0,17 6,47 114 6 5 5 25 Soltau-Fallingbostel 2,46 2,84 137 8 6 6 26 Stade 0,41 10,00 84 5 6 6 27 Uelzen 7,76 3,53 214 14 7 6 28 Verden -1,45 3,44 107 11 10 10 29 Delmenhorst -4,88 4,31 8 1 13 12 30 Emden -14,74 8,19 4 0 0 9 31 Oldenburg -6,84 4,12 103 15 15 14 32 Osnabrück -9,19 -3,12 290 21 7 7 33 Wilhelmshaven -8,55 10,30 10 0 0 6 34 Ammerland -8,99 6,28 76 4 5 6 35 Aurich -13,11 9,42 111 15 14 13 36 Cloppenburg -9,78 2,84 110 8 7 7 37 Emsland -13,60 1,07 242 21 9 8 38 Friesland -9,10 9,32 15 1 7 9 39 Bentheim -15,95 -1,46 36 1 3 4 40 Leer -12,42 6,67 39 2 5 6 41 Vechta -8,12 0,20 83 2 2 3 42 Wesermarsch -6,54 7,55 45 9 20 18 43 Wittmund -10,76 10,10 53 4 8 8 Sum 5365 706 Median (in %) 8,2 9,0 Figure 6 is a choropleth map of the empirical Bayes smoothed period prevalences from 43 regions in the study area. The cut points of the grey scale shading are again the 5%, 50% and 95% quantiles of the empirical distribution or the smoothed data. The period prevalences range from 3% to 51% with the median at 9%. Figure 6 Choropleth map of Bayesian smoothed prevalences in Lower Saxony. Choropleth map of the empirical Bayesian smoothed period prevalences from 43 regions in Lower Saxony. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. The map indicates high period prevalences in the south and north of Lower Saxony. Extraordinarily high prevalences were observed in the southern regions, which indicate the presence of a positive disease cluster that is identified by use of the spatial scan statistic [4]. Figure 7 shows the corresponding isopleth map resulting from universal kriging, with overlaid isolines. The isopleth map gives an impression of gradual changes instead of jumps at the regional borders. Furthermore, the missing region of Bremen in the central north of Lower Saxony has also been supplied with predicted values. Figure 7 Isopleth map from kriging the smoothed prevalences in Lower Saxony. Isopleth map with overlaid isolines of the kriging interpolated choropleth map in Figure 6. Isolines are at the 5%, 50% and 95% quantiles of the empirical distribution of the empirical Bayesian smoothed regional period prevalences. Figure 6 points out the presence of heterogeneity in the spatial mean. Here, universal kriging is based on the assumption that the spatial mean function of the data could be represented by an incomplete quadratic polynomial in the spatial co-ordinates. Other potential explanatory variables are not at hand. Let s = (x, y)' denote an arbitrary location in the study region, where x and y are the co-ordinates to the east and north. By inspection of the variogram cloud [12] based on trend residuals, two observations (Sites 3 and 13) were identified as outliers and removed from the structure analysis. In the second step of an iterated modelling approach then the trend polynomial fitted by ordinary least squares (OLSE) is given by μ(s) = β0 + β1y + β2y2, with , , . Instead of OLSE one could use iterated WLSE, but iteration may lead to biased estimates. The residuals of the trend surface fit to the empirical Bayes smoothed regional period prevalences were then used to model a spherical semivariogram by weighted least squares estimation of the robustly estimated empirical semivariogram. Figure 8 shows the empirical semivariogram as well as the fitted model for both, the detrended and trend contaminated data (sill = 0.0039, range = 5.29 and sill = 0.0047, range = 7.83 respectively), to indicate the benefit from the trend model as measured by the 20% decrease of the sill value. Figure 8 Empirical semivariograms and fitted exponential models from detrended and trend-contaminated smoothed prevalences. Robust empirical semivariograms of the detrended (black dot) and trend-contaminated data (circle) along with the WLSE fitted spherical models for the detrended (solid line) and the trend-contaminated (dashed line) data with observations 3 and 13 removed. The normal probability plot of the kriging cross-validation residuals in Figure 9 draws attention to the presence of three or four outliers (Observations 3, 8 and 13 and possibly 15). Observation 3 and 15 were previously identified as extremes and excluded from the analysis. Regions 8 and 13 are the nearest neighbours of Region 15. All three are part of a positive spatial cluster [4], a spatial structure that could not be captured by the spatial linear model used for universal kriging. However, the general appearance of the data is Gaussian. This in turn justifies the appropriateness of the modelling and prediction approach based on the empirical Bayesian smoothed regional data. Figure 9 Normal probability plot of cross-validation residuals from kriging smoothed prevalences. Normal probability plot of the cross-validation residuals from kriging the empirical Bayesian smoothed regional period prevalences. Observations 3, 8 and 13 may be spatial outliers with respect to the model based on the data set with observations 3 and 15 removed. Regions 8 and 13 are nearest neighbours of region 15, all three of which are part of a positive cluster. Geostatistical modelling of the empirical Bayesian smoothed regional prevalences allows the calculation of an error map representing the kriging standard errors. The darker the error map in Figure 10 the larger are the kriging standard errors, which range up from 0 to 0.06. Figure 10 Error map. Error map based on the universal kriging standard errors. Summary and discussion In this paper two sophisticated statistical methods were combined to solve an open problem in disease mapping. Mapping regional data using choropleth maps holds on to several problems that are overcome by isopleth mapping. To interpolate the regional, i.e. spatially discrete information, the geostatistical method of kriging is used here. Kriging requires variance homogeneous data. However, regional risk estimates are generally based on varying sample sizes and consequently turn up spatially varying standard errors. Therefore the spatial risk estimates are smoothed using linear empirical Bayes estimation. By borrowing-strength-from-the-ensemble, the impact of outliers is reduced and standard errors are stabilized over space. This cannot achieve variance homogeneity entirely nor could the tendency be shown analytically. Bootstrapping methods for spatially dependent data [18] promise an empirical justification and will be a source for future research. The purpose of exploratory disease mapping is to provide insight, as opposed to precise estimates of location, spread or trends [19]. Emphasise shall be on easy and intuitive statistical mapping methods. The method proposed here for exploratory disease mapping is on one hand based on empirical Bayesian estimation for smoothing which is related to internal standardization in epidemiology [[9], p. 260] and could thus be viewed as a natural choice for adjusting the data for spatially varying variances. On the other hand, the kriging predictions are weighted moving averages, where weights are chosen with respect to the spatial autocorrelation structure exhibited by the sample data. This concept is easily communicable to map users. Only monochrome colours or shades of grey should be used along with isopleth mapping [36], as has been done here in order to avoid the natural preference of the human eye for bright colours and in deference to colour-blind map users. Kriging is known to be a smoothing method and it may be argued that the proposed mapping method results in double or over-smoothing. But here kriging is based on a semivariogram model without nugget effect, which is known to be a direct interpolation method [12]. Therefore the risk map shows the Bayesian smoothed regional risks in the respective regional centres. The proposed disease mapping approach is a two-step procedure. The corresponding error map (Figure 10), however, neglects the error from the first step, i.e. the smoothing step. This is in line with error maps used along with similar sandwich predictors obtained via median polish kriging [3,12]. Thus the error map is useful to investigate the predictive performance of the kriging step but less useful for analytical inferences. The empirical Bayes method as described here does not take into account spatial autocorrelation, but a modification to overcome this lack has been proposed [29]. The method described thus far is called global smoothing, because the Bayes estimates are shrunk towards the global mean of all regions. The modification consists of using the local mean based on neighbouring regions instead of shrinking the estimates, which is called local smoothing. In any case, a simulation-based evaluation of a wide range of estimation methods [26] shows that the global smoother performs better overall than the local smoother. (This result may be related to the fact, that also regions with higher or lower population density cluster into urbanized regions and rural surroundings.) Only the full Bayesian approach to hierarchical modelling of regional disease data [5] outperforms the global smoother. But this modelling approach is certainly not suited for exploratory work. Conclusions Interpolation of the regional disease risk estimates overcomes the areal bias and related problems of choropleth disease mapping. Consequently, isopleth maps are easier to read and interpret than choropleth maps. The geostatistical method of kriging is appropriate for this task when based on linear empirical Bayesian smoothed data. Unlike non-parametric and mathematical interpolation methods, the spatial model underlying the exploratory spatial risk map offers ways of interpretation. Finally, the proposed concept for exploratory spatial risk mapping is easily communicable to map users. The Bayesian smoothing estimator is related to internal standardization in epidemiology. Also kriging can be viewed as weighted moving averaging. ==== Refs Berke O Estimation and prediction in the spatial linear model Water, Air, and Soil Pollution 1999 110 215 237 10.1023/A:1005035509922 Berke O Modified median polish kriging and its application to the Wolfcamp-Aquifer data Environmetrics 2001 12 731 748 10.1002/env.495.abs Berke O Choropleth mapping of regional count data of Echinococcus multilocularis among red foxes in Lower Saxony, Germany Preventive Veterinary Medicine 2001 52 119 131 11679170 10.1016/S0167-5877(01)00246-X Berke O von Keyserlingk M Broll S Kreienbrock L Zum Vorkommen von Echinococcus multilocularis bei Rotfüchsen in Niedersachsen: Identifikation eines Hochrisikogebietes mit Methoden der räumlichen epidemiologischen Clusteranalyse Berl Munch Tierarztl Wochenschr 2002 115 428 434 12481649 Besag J York J Mollié A Bayesian image restoration with applications in spatial statistics (with discussion) Annals of the Institute of Statistical Mathematics 1991 43 1 59 Bithell J An application of density estimation to geographical epidemiology Statistics in Medicine 1990 9 691 701 2218172 Bithell J A classification of disease mapping methods Statistics in Medicine 2000 19 2203 2215 10960848 10.1002/1097-0258(20000915/30)19:17/18<2203::AID-SIM564>3.0.CO;2-U Brillinger DR Spatio-temporal modelling of spatially aggregate birth data Survey Methodology 1990 16 255 269 Carlin BP Louis TA Bayes and Empirical Bayes Methods for Data Analysis 2000 2 London, Chapman and Hall Carrat F Valleron AJ Epidemiologic mapping using the "kriging" method: Application to an influenza-like epidemic in France American Journal of Epidemiology 1992 135 1293 1300 1626546 Clayton D Kaldor J Empirical Bayes estimates of age-standardized relative risks for use in disease mapping Biometrics 1987 43 671 681 3663823 Cressie N Statistics for Spatial Data 1993 rev New York, Wiley Cressie N Read TRC Spatial Data analysis of regional counts Biometrical Journal 1989 31 699 719 Diggle PJ Elliot P, Wakefield JC, Best NG, Briggs DJ Overview of statistical methods for disease mapping and its relationship to cluster detection Spatial Epidemiology: Methods and Applications 2000 Oxford, Oxford University Press 87 103 Diggle PJ Tawn JA Moyeed RA Model-based geostatistics (with discussion) Applied Statistics 1998 47 299 350 10.1111/1467-9876.00113 Eckert J Gemmell M Meslin FX Pawlowski Z WHO / OIE Manual on Echinococcosis in Humans and Animals: a Public Health Problem of Global Concern 2001 Paris, OIE Elliot P Wakefield JC Best NG Briggs DJ Spatial Epidemiology: Methods and Applications 2000 Oxford, Oxford University Press Garcia-Soidan PH Hall P On sample re-use methods for spatial data Biometrics 1997 53 273 281 Kafadar K Simultaneous smoothing and adjusting mortality rates in U.S. counties: melanoma in white females and white males Statistics in Medicine 1999 18 3167 3188 10602143 10.1002/(SICI)1097-0258(19991215)18:23<3167::AID-SIM308>3.0.CO;2-N Kellsall J Diggle PJ Non-parametric estimation of spatial variation in risk Statistics in Medicine 1995 14 2335 2342 8711273 Kelsall J Wakefield J Modelling spatial variation in disease risk: a geostatistical approach Journal of the American Statistical Association 2002 97 692 701 10.1198/016214502388618438 Kulldorff M A spatial scan statistic Communications in Statistics: Theory and Methods 1997 26 1481 1496 Lajaunie C Local risk estimation for a rare non contagious disease based on observed frequencies Note N-36/91/G Fotainebleau: Centre de Géostatistique, École des Mines de Paris 1991 Lawson AB Statistical Methods in Spatial Epidemiology 2001 Chichester, Wiley Lawson AB Tutorial in biostatistics: disease map reconstruction Statistics in Medicine 2001 20 2183 2203 11439429 10.1002/sim.933 Lawson AB Biggeri A Böhning D Lesaffre E Viel JF Clark A Schlattmann P Divino F Disease mapping models: an empirical evaluation Statistics in Medicine 2000 19 2217 2242 10960849 10.1002/1097-0258(20000915/30)19:17/18<2217::AID-SIM565>3.0.CO;2-E Lawson AB Clark A Spatial mixture relative risk models applied to disease mapping Statistics in Medicine 2002 21 359 370 11813223 10.1002/sim.1022 Lawson AB Williams F Application of extraction mapping in environmental epidemiology Statistics in Medicine 1993 12 1249 1258 8210824 Marshall R Mapping disease and mortality rates using empirical Bayes estimators Applied Statistics 1991 40 283 294 12157989 Martuzzi M Elliott P Empirical Bayes estimation of small area prevalence of non-rare conditions Statistics in Medicine 1996 15 1867 1873 8888479 10.1002/(SICI)1097-0258(19960915)15:17<1867::AID-SIM398>3.3.CO;2-U McNeill L Interpolation and smoothing of binomial data for the Southern African Bird Atlas Project South African Statistical Journal 1991 25 129 136 Morris CN Parametric empirical Bayes inference: theory and applications Journal of the American Statistical Association 1983 78 47 55 Müller HG Stadtmüller U Tabnak F Spatial smoothing of geographically aggregated data, with application to the construction of incidence maps Journal of the American Statistical Association 1997 92 61 71 Oliver MA Muir KR Webster R Parkes SE Cameron AH Stevens MCG Mann JR A geostatistical approach to the analysis of pattern in rare disease Journal of Public Health Medicine 1992 14 280 289 1419206 Oliver MA Webster R Lajaunie C Muir KR Binomial cokriging for estimating and mapping the risk of childhood cancer Journal of Mathematics Applied in Medicine and Biology 1998 15 279 297 Smans M Estève J Elliott P, Cuzick J, English D, Stern R Practical approaches to disease mapping Geographical and Environmental Epidemiology: Methods for Small-Area Studies 1992 Oxford, Oxford University Press 141 150 Wackernagel H Multivariate Geostatistics 1998 2 Berlin, Springer Webster R Oliver MA Muir KR Mann JR Kriging the local risk of a rare disease from a register of diagnoses Geographical Analysis 1994 26 168 185

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==== Front Comp HepatolComparative Hepatology1476-5926BioMed Central London 1476-5926-3-61533933310.1186/1476-5926-3-6ResearchSodium nitroprusside and peroxynitrite effect on hepatic DNases: an in vitro and in vivo study Kocic Gordana [email protected] Dusica [email protected] Radmila [email protected] Goran [email protected] Tatjana [email protected] Ivana [email protected] Tatjana [email protected] Radivoj [email protected] Dusan [email protected] Institute of Biochemistry, Medical Faculty University of Nis, Serbia and Montenegro2 Institute of Chemistry, Medical Faculty University of Nis, Serbia and Montenegro3 Clinic for Endocrinology, Faculty of Medicine University of Nis, Serbia and Montenegro2004 31 8 2004 3 6 6 17 11 2003 31 8 2004 Copyright © 2004 Kocic et al; licensee BioMed Central Ltd.2004Kocic et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mg2+- and caspase-dependent) and acid-DNase. The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite. Results The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after in vivo treatment with either SNP or peroxynitrite. A difference observed between the in vivo and in vitro effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased. Conclusion Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis. ==== Body Background In its response to tissue damage and inflammation induced by a variety of xenobiotics, endotoxins and disease states (such as viral hepatitis), post-ischemic and regenerative injury, the liver produces a large quantity of nitric oxide (NO). Nearly all cell types in liver tissue, including hepatocytes, Kupffer cells, stellate cells and endothelial cells, have the capacity for generating NO. It has been documented that NO is capable of promoting apoptosis in a number of different cell types, generally classified as cGMP-dependent or cGMP-independent [1-4]. The potential of chemical NO donor sodium nitroprusside (SNP) to induce apoptosis directly from NO liberation has been established in vitro [5]. The fact that NO is capable of triggering apoptosis is consistent with its ability to induce DNA damage, the inhibition of DNA synthesis and cell cycle arrest [6,7]. The reaction product formed between NO• and superoxide [i.e., peroxynitrite (ONOO-)] plays a critical role in the induction of inflammatory reaction and apoptosis, but is also associated with tumor promotion and/or progression. Potentially toxic levels of peroxynitrite can be achieved whenever NO• and O2.- production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO• and O2.- concentration [8]. Apoptosis, frequently termed "programmed cell death", is the form of cell death that occurs in normal liver in the course of its development and organogenesis, and in adult liver during the renewal of hepatocytes. In addition, apoptosis can be triggered by several hepatotropic viruses and toxic drugs, as well as in various liver diseases and experimental liver conditions such as hepatic allograft rejection. Degradation of the nuclear DNA, a common phenomenon observed in many organisms throughout the evolutionary scale, is one of the best-characterized biochemical features of apoptotic cell death. It has been established that the cell undergoes epigenetic reprogramming in the D1 phase of programmed cell death, the result of which is the activation of double-stranded DNA fragmentation in the F phase during which the nuclear morphology dramatically changes [9]. The cleavage of DNA may have a protective function in that it reduces the likelihood for genes in a potentially active site to be transferred from dying cells to the nuclei of viable neighboring cells. It is possible that various endonucleases exert a DNA degrading activity, as well as that many proteins can receive DNA degrading properties upon change of pH conditions [11]. The most important aspect of apoptosis is the universal property of some proteins to exert a dual function: the protection against proteolysis and the maintenance of the structure and function of normal cells. Being free from the inhibitory complex, however, these proteins may also contribute to protein or chromatin cleavage during apoptosis [12,13]. Changes in DNA degradation may lead to the pathogenesis in various disorders, such as liver cancer [14,15]. On the basis of the pharmacological data supporting the critical role of NO and peroxynitrite in apoptosis, current research studies have evaluated the activity of alkaline and acid DNase during the administration of SNP or of peroxynitrite, as well as the changes in numerous susceptible parameters of nitrosative stress, including SH group oxidation, carbonyl group formation, lipid peroxidation and DNA modification. An assay of enzyme activity was performed using liver tissue after in vivo administration and in vitro treatment of isolated rat hepatocytes or purified commercial enzymes either with SNP or authentic peroxynitrite. Results There are few data concerning the in vivo susceptibility of liver tissue to NO donor SNP and authentic peroxynitrite. A considerable attention has been paid to the establishment of in vivo tolerability and to the markers of apoptotic effects. Both NO and peroxynitrite can directly react with aromatic and sulfhydryl nucleophiles and nitrate aromatic residues. Sulfhydryl groups oxidation was documented in the plasma and, almost equally, in liver tissue. Peroxynitrite administration led to a more pronounced decrease in the concentration of other plasma free radical scavengers such as uric acid, and was followed by an increase in plasma nitrate concentration (Tables 1 and 2). According to the data suggesting that peroxynitrite decomposes rapidly to OH• and NO2 •-like species at physiological pH, it was assumed that the carbonyl groups and the lipid peroxidation product [i.e., malondyaldehyde (MDA)] may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous in vitro observations [16]. Plasma alanine aminotranferase (ALT) activity, used as the standard liver functional test, decreased in the peroxynitrite-treated group (Table 1). The activity of liver alkaline DNase increased and that of acid DNase decreased after in vivo treatment with either SNP or peroxynitrite (Table 2). Table 1 Plasma levels of investigated parameters of Sprague-Dawley rats after in vivo treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. Parameters Control SNP Peroxynitrite Lipid peroxidation (MDA) (μmol/l) 7.10 (0.86) 6.78 (2.11) 6.64 (2.85) SH groups (μmol/l) 180.47 (36.08) 150.84 (4.78) 110.40 (11.96)* NO2+NO3 (μmol/l) 45.47 (18.80) 66.82 (4.12) 81.94 (10.18) Uric acid (μmol/l) 64.82 (4.18) 22.34 (8.32) 17.50 (3.21)* Carbonyl groups (μmol/g protein) 179.59 (28.50) 144.90 (67.40) 169.06 (14.88) Nitrotyrosine (nmol/g protein) 96.02 (26.53) 136.17 (27.47) 135.11 (4.64) ALT (U/l) 31.9 (4.15) 34.44 (4.96) 28.5 (5.15) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated in bolus in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. Asterisk: significantly different from the control (p < 0.05). Table 2 Liver levels of investigated parameters of Sprague-Dawley rats after in vivo treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. Parameters Control SNP Peroxynitrite Lipid peroxidation (MDA) (μmol/g protein) 54.42 (8.68) 52.5 (4.34) 58.2 (6.11) SH groups (μmol/g protein) 636.04 (74.58) 538.67 (82.87) 505.52 (41.43) NO2+NO3 (μmol/g protein) 6.15 (2.91) 6.04 (2.61) 5.74 (1.03) Carbonyl groups (μmol/g protein) 4.14 (0.13) 4.25 (0.12) 4.20 (0.26) Nitrotyrosine (nmol/g protein) 10.26 (2.62) 11.40 (1.24) 16.62 (3.58) DNA (mg/g tissue) 10.71 (1.75) 12.65 (1.01) 12.15 (1.25) DNA fragmentation (%) 11.99 (0.33) 13.21 (1.43) 13.0 (0.89) Alkaline-DNase (U/g protein) 11.95 (0.57) 15.30 (0.72) 16.45 (1.04) Acid-DNase (U/g protein) 15.28 (3.42) 12.34 (0.35) 9.34 (0.76) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated in bolus in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. The ultraviolet spectra of DNA were obtained by spectrophotometric scanning between 230–500 nm on a scan detecting system. According to the data by Yermilov et al. [17], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. In the scan analysis (Figure 1), the peak was between 390 and 410 nm with a maximum absorbance at 405 nm in alkaline conditions (DNA extract was adjusted to pH 10). This peak may correspond to the formation of nitro-derivatives, most probably of 8-nitroguanine. The nitroguanine peak at 405 nm was particularly apparent in peroxynitrite-treated samples. Figure 1 The peak appearance of isolated liver DNA. The extraction oftissue DNA was performed according to the method of Wannemacher et al. [50], modified by Setaro & Morley [51], with the protein and nucleic acid precipitation by using ice-cold trichloroacetic acid after lipid extraction. DNA was separated from proteins by hydrolisis of resulting pellet at 96 ± 1°C for 45 min. Samples were analyzed for DNA concentration by ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes, monitored by using Beckman spectrophotometer. On the basis of the data obtained by Yermilov et al. [13], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. The peak appearance was between 390 and 410 nm with the maximum absorbance at 405 nm, obtained in alkaline conditions (DNA extract was adjusted to pH 10). In vivo administration of SNP or peroxynitrite tended to increase the rate of DNA fragmentation, but it was not statistically significant. The rate was estimated according to the percentage of DNA resisting centrifugation at 27 000 g (Table 2). After in vitro exposure of isolated hepatocytes to SNP or peroxynitrite, the activity of both alkaline and acid DNase decreased in a dose-dependent fashion (Figure 2). During in vitro incubation of purified enzymes DNase I and DNase II with SNP or peroxynitrite, a dose-dependent decrease of enzyme activity was also documented (Figure 3). Figure 2 The activity of alkaline and acid DNase after in vitro treatment of isolated hepatocytes with NO donor (SNP) or peroxynitrite. The isolation of hepatocytes was done according to the method already published [42], by using a 1% collagenase dissolved in RPMI 1640 medium. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 108cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given in vitro concentrations were calculated according to the literature data [38]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [43] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein. Data (n = 8) in graph is putted as: Mean + SD. Figure 3 The activity of commercial DNase I and DNase II after in vitro treatment with NO donor (SNP) or peroxynitrite. Purified enzymes DNase I (E.C. 3.1.21.1) and DNase II (E.C. 3.1.22.1) were dissolved in physiological saline solution. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 108 cells/ml. They were divided into seven groups (each comprising 8 samples) exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol). The reducing agent (cysteine 1 mmol) was added to SNP to induce in vitro NO release [39]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [43] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001/min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Data (n = 8) in graph is putted as: Mean + SD. Discussion NO•, a free radical gaseous molecule is one of the simplest compounds found to be continuously produced in humans and animals. It can be derived from L-arginine through the enzyme nitric oxide synthase (NOS) and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis [18-20]. Peroxynitrite is a strong, relatively long-lived oxidant with a half-life of approximately 0.5–1 s under physiological conditions. Our study confirmed that in both plasma and liver tissue peroxynitrite causes a rapid oxidation of sulfhydryl groups and thioethers, as well as the nitration and hydroxylation of aromatic compounds (Tables 1 and 2). A chronic exposure of hepatocytes to reactive nitrogen species exhibits a cytotoxic and cytostatic activity leading to functional and morphological alterations [8,21]. Cell death after exposure to different NO-donors such as SNP has been to date established through the expression of tumor suppressor gene p53 and pro-apoptotic genes such as bax, cyclin-dependent kinase inhibitor p21, the inhibited expression of anti-apoptotic protein bcl-2, the inhibited NF-κB binding activity, ERK and p-38-dependent cytochrome c release, and caspase-3 activation [22-24]. In contrast, the anti-apoptotic effects of NO may be mediated through the mechanisms such as blockade of the recruitment of pro-caspase-9 to the Apaf-1 apoptosome, stimulation of c-GMP-dependent protein kinase, control of mitochondrial permeability transition, induction of the heat shock protein HSP 70, and interaction with the ceramide pathway [25,26]. The prolonged damage of p53 gene by peroxynitrite has been associated with tumor formation. Recent results by Vincent and Maiese [3] indicate that NO donor SNP (at 300 μmol concentration) is capable of inducing strong apoptotic effects via DNA fragmentation and induction of Mg2+-dependent endonuclease activity in the culture of neuronal cells. In our in vivo study (Table 2), the activity of alkaline DNase increased within 24 h after exposure to SNP (achieving approximately a similar blood concentration of about 250 μmol) or to authentic peroxynitrite. Several molecules involved in nuclear DNA fragmentation have been detected and characterized based on their ionic sensitivity. Besides the presence of constitutive Ca2+/Mg2+-dependent endonucleases, a great deal of endonuclease activity within a 7.2–8.0 pH range most probably represents the inducible form of DNase. The molecular weights of the constitutive (NO-independent) and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5–8.0). A likely conclusion is that Mg2+-dependent endonuclease seems to be a result of de novo synthesized or the pre-existing Ca2+/Mg2+-dependent endonuclease activation. Up to now, several Mg2+- or Ca2+/Mg2+-dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5–9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD [27,28]. DNase gamma has been documented as a critical component of apoptotic machinery, in that it cleaves the chromosomal DNA into nucleosomal units, thus leading to DNA ladder formation [29]. The alkaline DNase, active only during apoptosis, has been documented to be inherent to cyclophilins (A, B and C) as well, irrespective of their protein folding (peptidylprolyl cis-trans-isomerase) activity. All of them have the ability to degrade the supercoiled, single stranded and double stranded DNA [30,31]. Besides alkaline DNases, the cation-independent endonuclease with an optimum activity at pH 5, known as acid or DNase II, was identified. One leucocyte elastase inhibitor (LEI) can also exert an acid DNase activity after post-translational modification through the proteolytic cleavage [32]. The specific involvement of DNase II in physiological nuclear degradation during apoptosis could not be excluded upon decrease of intracellular pH values below 7 with a proton ionophore. Three potential N-nitrosylation sites are important for DNase II regulation [32,33]. Since our experimental data indicated a decrease in acid DNase activity 24 h after exposure to SNP or peroxynitrite (Table 2), the inhibition of DNase II may be explained by the nitrosylation of its susceptible sites. Indeed, when isolated hepatocytes were exposed to SNP or peroxynitrite for 1 h, a dose-dependent inhibition of DNase II was also documented (Fig. 2). The same result was obtained after exposure of purified enzyme to SNP (in the presence of the reducing agent cysteine 1 mmol) or peroxynitrite (Fig. 3). The formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack [34,35]. A highly potential mutagenic product 8-nitroguanine can be depurinated yielding apurinic sites capable of inducing GC→TA transversions, GC→CG transversions and deletions [17,36]. The appearance of the nitroguanine peak during the scan analysis of purified DNA at 405 nm was documented in our study (Fig. 1). The rate of DNA fragmentation tended to be increased, but the difference was not significant (Table 2). Conclusions In vivo administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis. Methods Chemicals SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique [37] and its concentration was monitored in alkaline solution before use in each experiment by measuring the extinction coefficient at 302 nm [38]. All other chemicals were of the highest purity range. In vivo study Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated in bolus in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their in vivo tolerability and in vitro ability to induce apoptotic effects [39,40]. The calculation of peroxynitrite intra-arterial concentration (6 nmol) was done according to its biological half-life of about 0.6 s, cardiac output of 40 ml/min/100 g and circulating volume of 20 ml and 250 g of rat BW [41]. The corresponding control group received physiological saline solution in the same volume. The rats were killed 24 h afterwards, under the same anesthesia. Blood was collected from the abdominal aorta and livers were quickly removed, frozen and homogenised on ice. Isolation of hepatocytes The isolation of hepatocytes was done according to a method already published [42], by using a 1% collagenase dissolved in RPMI 1640 medium. Collagenase was inhibited by using 10% FCS and cells were washed twice in physiological saline solution. Hepatocytes were isolated from 8 Male Sprague-Dawley rats. They were dissolved in a physiological saline solution in a concentration approximately 108 cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given in vitro concentrations were calculated according to the literature data [38]. Purified enzymes DNase I and DNase II were dissolved in physiological saline solution, exposed to the same concentrations of SNP and peroxynitrite, except that the reducing agent (cysteine 1 mmol) was added to SNP to induce in vitro NO release [39]. Methods for alkaline and acid-DNase The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. [43] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein, for tissue and cell samples. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001 / min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Extraction of DNA and proteins The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al. [44] modified by Setaro & Morley [45] by protein and nucleic acid precipitation using ice-cold trichloroacetic acid (TCA), 0.6 N, after lipid extraction. RNA and DNA were isolated by using cold 60% perchloric acid (PCA). DNA was separated from proteins by hydrolysis of resulting pellet at 96 ± 1°C for 45 min after adding 0.5 N PCA. Tissue protein content was measured according to the Lowry et al. procedure [46]. Samples were analysed for DNA concentration by an ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes monitored by using Beckman DU 530 spectrophotometer. Protein carbonyls and protein nitrotyrosine were measured in plasma proteins and the remaining protein pellet according to the method of Oliver et al. [47] modified by Tien et al. [48]. DNA fragmentation assay was performed according to the method of Jones et al. [49] based on the percentage of DNA resisting centrifugation at 27 000 g for 20 min. The proportion is expressed as percentage of the total DNA in the uncentrifugated sample. Protein carbonyls were quantified by spectrophotometric measurement of their 2,4 dinitrophenylhydrazone derivatives (ε 370 nm = 22000 M-1 cm-1). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [50]. Plasma and tissue lipid peroxidation product MDA was measured according to the method of Ohkava et al. [51]. Nitrates were measured according to the method of Navarro-Gonzales et al. [52]. Plasma uric acid and ALT were measured using the Synchron analyzer. Statistics Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups. Authors' contributions GK carried out the in vivo and in vitro experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during in vivo and in vitro experiments and did the graphical presentation. DS performed statistical analysis and assisted during in vivo experiments. DP and RK assisted during in vitro experiments and participated in the design of the study. All the authors read and approved the final manuscript. ==== Refs Searle J Harmon BV Bishop CJ Kerr JFR The significance of cell death by apoptosis in hepatobiliary disease J Gastroenterol Hepatol 1987 2 77 96 Laskin JD Heck DE Gardner CR Laskin DL Prooxidant and antioxidant functions of nitric oxide in liver toxicity Antioxid Redox Signal 2001 3 261 271 11396480 10.1089/152308601300185214 Vincent AM Maiese K Nitric oxide induction of neuronal endonuclease activity in programmed cell death Exp Cell Res 1999 246 290 300 9925743 10.1006/excr.1998.4282 Nishio E Fukushima K Shiozaki M Watanabe Y Nitric oxide donor SNAP induces apoptosis in smooth muscle cells through cGMP-independent mechanism Biochem Biophys Res Commun 1996 221 163 168 8660329 10.1006/bbrc.1996.0563 Feldmann G Liver apoptosis J Hepatol 1997 26 1 11 9204404 10.1016/S0168-8278(97)80491-6 Wink DA Kasprzak KS Maragos CM Elespuru RK Misra M Dunams TM Cebula A Koch WH Andrews AW Allen JS Keefer LK DNA deaminating ability and genotoxicity of nitric oxide and its progenitors Science 1991 254 1001 1003 1948068 Nguyen T Brunson D Crespi CL Penman BW Wishnok JS Tannenbaum SR DNA damage and mutation in human cells exposed to nitric oxide in vitro Proc Natl Acad Sci USA 1992 89 3030 3034 1557408 Radi R Beckman JS Bush KM Freeman BA Peroxynitrite oxidation of sulfhydryls: The cytotoxic potential of superoxide and nitric oxide J Biol Chem 1991 266 4244 4250 1847917 Kung AL Zetterberg A Sherwood SW Schimke RT Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation Cancer Res 1990 50 7307 7317 2121336 Furuya Y Isaacs JT Differential gene regulation during programmed death (apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation Endocrinology 1993 133 2660 2666 8243289 10.1210/en.133.6.2660 Arends MJ Morris RG Wyllie AH Apoptosis: The role of the endonuclease Am J Pathol 1990 136 593 608 2156431 Kumar S ICE-like proteases in apoptosis Trends Biochem Sci 1995 20 198 202 7610484 10.1016/S0968-0004(00)89007-6 Los M Van de Craen M Penning LC Schenk H Westendorp HM Baeuerle PA Dröge W Krammer PH Fiers W Schulze-Osthoff K Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis Nature 1995 375 81 83 7536901 10.1038/375081a0 Isaacs JT Role of programmed cell death in carcinogenesis Environ Health Perspect 1993 101 27 33 8013419 Raff M Cell suicide for beginners Nature 1998 396 119 122 9823889 10.1038/24055 Bernabe JC Tejedo JR Rincon P Cahuana GM Ramirey R Sobrino F Bedoya FJ Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation Exp Cell Res 2001 269 222 229 11570814 10.1006/excr.2001.5315 Yermilov V Rubio J Becchi M Friesen MD Pignatelli B Ohshima H Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro Carcinogenesis 1995 16 2045 2050 7554052 Geller DA Nussler AK Di Silvio M Lowenstein CJ Shapiro RA Wang SC Simmons RL Billiar TR Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes Proc Natl Acad Sci USA 1993 90 522 526 7678458 Cohen RA The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease Prog Cardiovasc Dis 1995 38 105 128 7568902 Serracino FI Mathie RT Nitric oxide and hepatic ischemia-reperfusion injury Hepatogastroenterology 2000 47 1722 1725 11149042 D'Ambrosio SM Gibson-D'Ambrosio RE Brady T Oberyszyn AS Robertson FM Mechanism of nitric oxide-induced cytotoxicity in normal human hepatocytes Environ Mol Mutagen 2001 37 46 54 11170241 10.1002/1098-2280(2001)37:1<46::AID-EM1005>3.0.CO;2-6 Pinsky DJ Aji W Szabolcs M Athan ES Liu Y Yang YM Kline RP Olson KE Cannon PJ Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocites in culture Am J Physiol 1999 277 H1189 H1199 10484441 Kolb JP Mechanisms involved in the pro- and anti-apoptotic role of NO in human leukemia Leukemia 2000 14 1685 1694 10995017 10.1038/sj.leu.2401896 Tsi CJ Chao Y Chen CW Lin WW Aurintricarboxylic acid protects against cell death caused by lipopolysaccharide in macrophages by decreasing inducible nitric oxide synthase induction via I kappa B kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase inhibition Mol Pharmacol 2002 62 90 101 12065759 10.1124/mol.62.1.90 Mannick JB Asano K Izum K Kieff E Stamler JS Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation Cell 1994 79 1137 1146 7528106 10.1016/0092-8674(94)90005-1 Genaro AM Hortelano S Alvarez A Martinez C Bosca L Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels J Clin Invest 1995 95 1884 1890 7706495 Enari MH Sakahira H Yokoyama K Okawa A Iwamatsu A Nagata S A caspase-activated DNase that degrades DNA during apoptosis and its inhibitor ICAD Nature 1998 391 43 50 9422506 10.1038/34112 Liu X Li P Widlak P Zou H Luo X Garrard WT Wang X The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis Proc Natl Acad Sci USA 1998 95 8461 8466 9671700 10.1073/pnas.95.15.8461 Nishimura K Tanuma S Presence of DNase gamma-like endonuclease in nuclei of neuronal differentiated PC12 cells Apoptosis 1998 3 97 103 14646507 10.1023/A:1009644924530 Montague JW Hughes FJ Cidlowski JA Native recombinant cyclophylins A, B and C degrade DNA independently of peptydyl-prolyl cis-trans-isomerase activity. Potential roles of cyclophylins in apoptosis J Biol Chem 1997 272 6677 6684 9045699 10.1074/jbc.272.10.6677 Nagata T Kishi H Liu QL Yoshino T Matsuda T Jin ZX Murayama K Tsukada K Muraguchi A Possible Involvement of Cyclophilin B and Caspase-Activated Deoxyribonuclease in the Induction of Chromosomal DNA Degradation in TCR-Stimulated Thymocytes J Immunol 2000 165 4281 4289 11035062 Torriglia A Chaudun E Chany-Fournier F Jeanny C Courtois CJ Counis YMF Involvement of DNase II in Nuclear Degeneration during Lens Cell Differentiation J Biol Chem 1995 270 28579 28585 7499373 10.1074/jbc.270.48.28579 Counis MF L-DNase II, a Molecule That Links Proteases and Endonucleases in Apoptosis, Derives from the Ubiquitous Serpin Leukocyte Elastase Inhibitor Mol Cell Biol 1998 18 3612 3619 9584202 Wu YC Stanfield GM Horvitz HR NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis Genes & Dev 2000 14 536 548 10716942 Epe B Ballmaier D Roussyn I Briviba K Sies H DNA damage by peroxynitrite characterized with DNA repair enzymes Nucl Acids Res 1996 24 4105 4110 8932358 10.1093/nar/24.21.4105 Zingarelli B O'Connor M Wong H Salzman AL Szabó C Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide J Immunol 1996 156 350 353 8598485 Szabo C Ohshima H DNA damage induced by peroxynitrite: subsequent biological effects Nitric Oxide 1997 1 373 385 9441908 10.1006/niox.1997.0143 Fici GJ Althaus JS Hall ED VonVoigtlander PF Protective effects of tirilazad mesylate in a cellular model of peroxynitrite toxicity Res Commun Mol Pathol Pharmacol 1996 91 357 371 8829775 Tuo J Wolff SP Loft S Poulsen HE Formation of nitrated and hydroxylated aromatic compounds from benzene and peroxynitrite, a possible mechanism of benzene genotoxicity Free Radic Res 1998 28 369 375 9684981 Bates JN Baker MT Guerra Harrison RJ Nitric oxide generation from nitroprusside by vascular tissue Biochem Pharmacol 1991 42 S157 S165 1768273 10.1016/0006-2952(91)90406-U Nossuli TO Hayward R Jensen D Scalia R Lefer AM Mechanism of cardioprotection by peroxynitrite in myocardial ischemia and reperfusion injury Am J Physiol 1998 275 H509 H526 9683439 Graves JE Lewis SJ Kooy NW Peroxynitrite-mediated vasorelaxation: evidence against the formation of circulating S-nitrosothiols Am J Physiol 1998 274 H1001 H1008 9530214 Kocic G Vlahovic P Pavlovic D Kocic R Jevtovic T Cvetkovic T Stojanovic I The possible importance of the cation-binding site for the oxidative modification of liver 5'-nucleotidase Arch Physiol Biochem 1998 106 91 99 9894865 10.1076/apab.106.2.91.4386 Bartholeyns J Peeters-Joris C Reychler H Baudhun P Hepatic nucleases 1. Method for the specific determination and characterization in rat liver Eur J Biochem 1975 57 205 211 240718 Wannemacher RW Banks WL Wunner WH Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of amino acid incorporation Anal Biochem 1965 11 320 326 5891456 Setaro F Morley CD A rapid colorimetric assay for DNA Anal Biochem 1977 81 467 471 907109 Lowry OH Rosenbrough NJ Farr AJ Randall RJ Protein measurement with the pholin phenol reagent J Biol Chem 1951 193 265 275 14907713 Oliver CN Ahn B Moerman EJ Goldstein S Stadtman ER Age-related changes in oxidized proteins J Biol Chem 1987 262 5488 5491 3571220 Tien M Berlett BS Levine RL Chock PB Stadtman ER Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentartion: pH dependence of carbonyl formation, tyrosine nitration and methionine oxidation Proc Natl Acad Sci USA 1999 96 7809 7814 10393903 10.1073/pnas.96.14.7809 Jones DP McConkey DJ Nicotera P Orrenius S Calcium-activated DNA fragmentation in rat liver nuclei J Biol Chem 1989 264 6398 6403 2703497 Ellman LG Tissue sulfhydryl groups Arch of Biochem Biophys 1959 82 70 77 13650640 Ohkava H Ohishi N Yagi K Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction Anal Biochem 1979 95 351 358 36810 Navarro-Gonzales JA Garcia-Benayas C Arenos J Semiautomated measurement of nitrate in biological fluids Clin Chem 1998 44 679 682 9510886

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==== Front Cardiovasc UltrasoundCardiovascular Ultrasound1476-7120BioMed Central London 1476-7120-2-151533101510.1186/1476-7120-2-15ReviewCombination of contrast with stress echocardiography: A practical guide to methods and interpretation Moir Stuart [email protected] Thomas H [email protected] Division of Medicine, University of Queensland, Brisbane, Australia2004 26 8 2004 2 15 15 16 7 2004 26 8 2004 Copyright © 2004 Moir and Marwick; licensee BioMed Central Ltd.2004Moir and Marwick; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contrast echocardiography has an established role for enhancement of the right heart Doppler signals, the detection of intra-cardiac shunts, and most recently for left ventricular cavity opacification (LVO). The use of intravenously administered micro-bubbles to traverse the myocardial microcirculation in order to outline myocardial viability and perfusion has been the source of research studies for a number of years. Despite the enthusiasm of investigators, myocardial contrast echocardiography (MCE) has not attained routine clinical use and LV opacification during stress has been less widely adopted than the data would support. The purpose of this review is to facilitate an understanding of the involved imaging technologies that have made this technique more feasible for clinical practice, and to guide its introduction into the practice of the non-expert user. ==== Body I. Imaging principles Micro-bubbles Two aspects of micro-bubbles are important – their gas content and the nature of their shell. Recently-approved micro-bubbles almost universally involve encapsulation of a high molecular weight gas, which improves the persistence of the bubble, optimising the number available in the left heart chambers. Air has a greater propensity to dissolve into solution and although currently unattractive because of loss of gas before arrival on the right side of the heart, better encapsulation may allow its resurgence – the benefit would be more rapid disappearance when the bubble bursts. The nature of the shell or surface modifying agent, which improves stability and prevents dissolution, may become important for new targeted imaging approaches. Interaction of micro-bubbles and ultrasound Even when optimal microbubble delivery to the myocardium is achieved with invasive coronary [1] or aortic root [2] injections, detection of reliable myocardial opacification using standard 2D imaging is difficult. Whilst this partly reflects difficulty distinguishing bright, grey scale echo signals from the myocardial tissue from those of micro-bubbles within the myocardial micro-circulation, the problem is multi-factorial and has been overcome by the development of contrast specific imaging modalities, which exploit the unique interaction between the ultrasound field and micro bubbles[3] to maximise the received contrast backscatter and minimise myocardial tissue backscatter. Micro-bubbles oscillate (expand and contract) in the ultrasound field. The pattern and nature of their oscillation, and thus the nature of the backscatter signal, differs, depending on the acoustic power of the transmitted ultrasound field, which is expressed on modern ultrasound machines as the mechanical index (MI). In general echocardiography, the amplitude of returning backscatter depends on the nature of the insonated structure, and is represented by brightness on the formed image. Most backscatter returns at the same frequency as the transmitted ultrasound (fundamental frequency). With very low mechanical index imaging (MI < 0.1), micro bubbles demonstrate linear oscillation, where the contraction and expansion of the micro bubble are equal. All returning micro bubble backscatter remains within the range of the frequency transmitted by the transducer (the fundamental frequency) – as for surrounding structures (Figure 1a). Figure 1 Interaction of micro-bubbles and ultrasound. Low mechanical index imaging (0.1 – 0.3), generates nonlinear oscillation of the micro bubble whereby expansion is greater than contraction. In addition to the usual backscatter of a particular amplitude within the range of the fundamental frequency, the bubbles also produce backscatter of (lesser) amplitude at harmonic frequencies [4,5]. For example scatter from a bubble interrogated by a 4 MHz pulse will be composed of 4 MHz and 8 MHz sound (Figure 1b). Tissues and surrounding structures do not produce harmonic backscatter when imaged under low power. When exposed to high mechanical index imaging (MI > 0.6, ie the MI used for standard imaging) the bubbles oscillate wildly and burst. Upon destruction, micro-bubbles produce a brief, high amplitude signal, with good resolution, which is rich in harmonic signals, containing backscatter at the second third and fourth harmonics etc (Figure 1c). The bubble dissipates gradually following destruction, depending upon the gas composition of the bubble, with air-based micro-bubbles dissipating much more rapidly. Importantly at high MI, tissues also produce harmonics. With standard fundamental imaging (high MI), myocardial micro-bubbles are destroyed continuously, causing apical swirling on LVO images and never replenishing the myocardium within the beam, and thus not returning significant myocardial backscatter. This exacerbates the underlying difficulty of differentiating on grey scale, between tissue backscatter and micro-bubble backscatter. Changing to standard harmonic imaging (processing only signals returned at the second harmonic of the fundamental frequency) significantly improves LVO, but offers little benefit for myocardial perfusion, with ongoing bubble destruction and sub-optimal differentiation between contrast and tissue, because both produce harmonics at high power. Contrast specific imaging modalities, with the aim of enhancing the contrast to tissue backscatter signal ratio (CTR) are required for optimal performance of a myocardial contrast study. Contrast specific imaging technology High Power Techniques – Intermittent Imaging Triggered harmonic imaging Porter demonstrated that intermittent high power imaging demonstrated significantly better myocardial opacification than continuous imaging, with the best opacification obtained using intermittent harmonic imaging [6]. During intermittent high power imaging, high energy ultrasound is transmitted at specified intermittent intervals, triggered to the ECG (eg once every 4 cardiac cycles; 1:4 triggering). The time between destructive pulses allows the micro-bubbles to replenish the myocardium. With each destructive pulse, high amplitude backscatter rich in harmonics is returned to the transducer, enabling static images of myocardial perfusion. Moreover, by incrementally increasing the triggering intervals (continuous → 1:1 → 1:2 → 1:4 → 1:8 etc), the rate of replenishment of the ultrasound beam over time can be assessed both qualitatively and quantitatively. The qualitative assessment of perfusion using intermittent harmonic imaging can be improved by digital subtraction of the myocardial signal, and optimized by colour coding techniques to allow better extraction of bubble signals [7] (Figure 2). The first human study validating myocardial perfusion assessment by MCE with SPECT used digital subtraction [8]. However, this approach requires careful superimposition of each frame, and although the analysis is becoming more automated, the processing is still performed off-line. Figure 2 Digital subtraction and colour coding of MCE images acquired using intermittent harmonic imaging. Harmonic Power Doppler Power Doppler technology is designed to detect motion of blood or tissue, and is used in myocardial contrast echo to overcome the need for off line digital subtraction [9]. As with traditional Doppler, two or more pulses are sent successively along each scan line of the image and the pairs of echo trains are compared for the presence or absence of a frequency shift (indicating movement). However, unlike traditional Doppler, power Doppler ignores the direction and velocity of the moving structure; if a frequency shift is detected, indicating motion of the structure, then colour is displayed as an overlay whose saturation is related to the amplitude of the echo which has moved. If no frequency shift (movement) is detected, no colour is displayed. This method is ideally suited to high mechanical index destructive imaging, as the first pulse destroys the myocardial micro-bubbles, generating a brief, high amplitude echo rich in harmonics. The second pulse finds that the bubbles have 'moved', and thus colour is overlaid on the echo image over the areas of myocardium that contained micro-bubbles. In an area with no micro bubbles, there is no 'movement' recognised and there is no colour overlay applied to that region. Note that the technique is not actually detecting movement of the bubbles in the circulation, but rather their destruction. This technique has been correlated in human subjects with SPECT[10] and has been used extensively in clinical trials (Figure 3 and additional file 1). Figure 3 Harmonic power Doppler imaging [see also additional file 1] The most important limitation of this technique is motion artefact from tissue, because tissue motion will be expressed like bubble destruction, potentially showing perfusion when none is present (a false negative). If Doppler frequency is increased, pulse separation is reduced, so tissue movement between pulses can be minimized. However, if the pulses are too close, not all the gas within the bubble will have dissipated before arrival of the next pulse, so the difference between pulses is reduced, possibly leading to false positive perfusion defects. Air-filled micro-bubbles are optimal for this technique because of rapid dissipation of the gas, allowing closely spaced pulses. Pulse Inversion Doppler Another grey scale high MI technique is pulse-inversion imaging whereby two beam mode pulses are sent in rapid succession into the myocardium. The second pulse sent is a mirror image of the first (i.e. 180° phase shift). The scanner processes the consecutive returning pulses by adding them together. Tissue generates a linear echo, thus the addition of one pulse to the other should cancel out to zero and no signal is generated. Micro-bubbles produce non-linear echo signals and the summation of returning pulses will not equal zero and a signal will be registered (Figure 4). Figure 4 Pulse inversion Doppler. Using this technique, processing can theoretically be limited only to signals generated by bubbles. As well as being a grey-scale technique, tissue motion artefacts are a major limitation, as movement of tissue also creates non-linear signals. Nevertheless the theory behind this technology has led to the development of successful real time imaging. Ultraharmonic imaging With the development of increased bandwidth transducers, another intermittent high MI technique has evolved to improve the contrast to tissue ratio of backscatter. While intermittent harmonic imaging demonstrates myocardial perfusion, it is limited by the presence of tissue signals at the second harmonic. However, bubble destruction causes backscatter at 3rd 4th and sub-harmonics. At these higher harmonics the tissue signal is negligible, thus processing only backscatter from these further harmonics enables selective enhancement of the contrast signal. Interestingly, current ultra-harmonic imaging involves processing signals from between the second and third harmonic. The strength of the high MI approaches are their sensitivity for the presence of contrast, because bubble destruction results in the highest amplitude backscatter. The disadvantages are that they lack simultaneous assessment of function, require reliable ECG triggering and image acquisition, and can be both technically challenging and time consuming (of particular importance for stress imaging). While the difficulty maintaining image position with long triggering intervals has been aided by the use of low MI localization images, respiratory movement is almost inevitable. Low Power Techniques – Real Time Imaging In recent years, technologies have been developed which so successfully exploit the non-linear responses of micro-bubbles, that even low amplitude micro-bubble backscatter can be isolated from tissue signals for processing. This allows continuous low power imaging to be performed, with limited bubble destruction, enabling simultaneous assessment of wall motion and perfusion in real time (although frame-rate is minimized in order to reduce bubble destruction) (See table 1). Hence low MI imaging is commonly known as real time imaging, The use of low MI has two major benefits; (1) The bubbles undergo stable non-linear oscillation emitting continuous fundamental and harmonic signals and (2) the tissues themselves do not generate harmonic signals at low MI. Like incremental triggered imaging, low MI imaging enables assessment of micro-bubble replenishment of the myocardium over time after bubble destruction. With low MI imaging, myocardial bubble destruction is achieved by transmission of a series of a high MI pulses ("flashes"), after which replenishment can be observed in real time (see figure 11). There are 2 major real time techniques. Figure 11 Destruction replenishment imaging with real time MCE – see text. Power pulse inversion imaging This technique combines the non-linear detection performance of pulse inversion with the motion discrimination capability of power Doppler. Multiple transmit pulses of alternating polarity are used, and Doppler signal processing techniques are applied to distinguish between bubble backscatter and backscatter from tissue. In a typical configuration, echos from a train of pulses are combined in such a way that signals from moving tissue are eliminated (see also figure 4). Power modulation imaging This method utilises the same signal subtraction principles, as PPI, with the transmitted pulses identical in phase but of different in amplitude or 'power' – hence power modulation, one impulse of full power, the other half that power. Echos reflected from stationery tissue are linear, thus if we subtract two times the lower power from full power, the signals should cancel out whereas a non-linear oscillation of micro-bubbles will generate a signal. In power modulation, fundamental imaging is most suited, because the tissue subtraction technique is so effective that the best signal to noise ratio from contrast to tissue is at the fundamental frequency. Both techniques have been validated in animals and utilised for qualitative assessment of perfusion in humans[11,12]. Bubble administration The decision regarding the use of a bolus or infusion for intravenous administration of micro-bubbles is dependant on a variety of factors, including the type of micro-bubble used, equipment and staff available and clinical indication for the test (LVO, qualitative perfusion or quantitative perfusion). For MCE, bubbles should be infused with the aim of LV opacification and adequate myocardial perfusion with minimal/no attenuation of the basal segments. The amount of contrast required for this varies from bubble to bubble, machine to machine and technique used (intermittent or continuous), as well as showing patient to patient variability. Ideally all studies, particularly those assessing perfusion would involve a continuous infusion of micro-bubbles, with a mechanically controlled infusion preferable to a manually controlled one (slow continuous injection). This enables establishment of a true steady state for optimal imaging and in particular quantification. II. Left ventricular opacification Accurate evaluation of regional and global left ventricular function by echocardiography is dependent on adequate endocardial border resolution. Using fundamental imaging, approximately 20% of resting echos demonstrate inadequate endocardial definition [13], defined as ≥ 2 segments not seen at baseline. While native tissue harmonic imaging enables better endocardial definition than standard fundamental imaging and reduces the number of patients with inadequate studies to 5–10%, contrast induced LVO still confers benefit over harmonic imaging [14]. The most challenging patients have obesity, chronic lung disease or chest wall deformities. Ventilated patients in intensive care also provide significant difficulties because of patient positioning and compliance. Techniques that enhance discrimination between myocardial tissue and the blood pool may therefore improve the clinical utility and diagnostic accuracy of echo. Left ventricular opacification (LVO) by contrast echo enhances this discrimination to better define the endocardial surface. Contemporary LVO involves administration of perflurocarbon micro-bubbles (which show superior duration of opacification and enhancement of endocardial definition compared with air filled bubbles [15]), and intermediate MI harmonic imaging (0.4–0.5) which allows continuous high frame rate imaging, results in reduced bubble destruction, leads to production of micro-bubble harmonic signals with minimal tissue harmonic production, enabling maximal discrimination between the opacified blood pool and myocardium (Figure 5 and additional files 2, 3, 4, and 5). Figure 5 Importance of machine settings for using contrast for LV opacification. (a) In this example, endocardial border definition is probably adequate with standard tissue harmonic imaging – [see additional file 2.] (b) The use of contrast for LVO with standard diagnostic harmonic imaging machine settings provides worse border definition in the lateral wall, and apical bubble destruction, illustrating the importance of appropriate machine settings – [see additional file 3]. Image (c) shows machine settings for myocardial perfusion imaging – this provides assessment of myocardial perfusion and wall motion, but the frame rate for WMA is 20–25 Hz and thus subtle WMA's could be missed – [see additional file 4]. Therefore for optimal assessment of WMA image (d) displays specific intermediate MI imaging at high frame rate designed specifically to enhance the endocardial/cavity border. Even in this example there is some apical swirling despite the focal zone set in the mid LV – [see also additional file 5]. LV volume measurement While there is clear evidence that contrast LVO can improve endocardial border definition, there are theoretical reasons why this may not necessarily translate into more accurate assessment of LV cavity size, volume and function. Attenuation artefacts sometimes obscure the endocardial surface, particularly in the basal segments and myocardial contrast may confuse distinction of the border between the blood pool and the endocardium. Most importantly, the use of any 2-D echo technique to assess left ventricular volumes and ejection fraction (with or without contrast), requires a standard imaging plane. Fore-shortened views or views not oriented through the centre of the ventricular cavity will lead to an underestimation of volume no matter how good the endocardial border definition [16]. Notwithstanding these potential technical limitations, contrast LVO using fundamental imaging improves the correlation between LV volumes obtained with echo and MRI [17]. In addition, accurate classification of systolic function by calculated ejection fraction has been improved from 70 to 94% by the use of contrast, with this improved accuracy almost exclusive to patients with poor baseline images. A subsequent study confirmed that, even using harmonic imaging, the measurement of LV volumes was optimised with contrast LVO, using electron beam CT as the standard of reference [18]. While the role of LVO for 3D imaging is currently undefined, it seems likely that this will be an important application of contrast echo (Figure 6 and additional files 6, 7, 8 and 9). Figure 6 Realtime 3D echocardiography without (a) – [additional file 6], and with contrast enhancement (b) [additional file 7]. There is clear benefit for LV border detection. [See also additional files 8 and 9 for real time 3-D movies without and with contrast respectively.] LVO and wall motion analysis Enhanced endocardial border definition can improve the accuracy and inter-observer agreement for assessment of regional wall motion at rest [19]. Compared with MRI, the number of segments visualized was clearly improved after contrast (86% visualised before contrast, 99% after contrast). In addition, identification of segments with abnormal wall motion improved 82% to 100%, with the clinical utility of the contrast being greatest at the lateral and anterior walls. Importantly, the inter-observer agreement for assessment of individual wall segments was significantly improved and contrast also improved intra-observer agreement for determination of normal versus abnormal wall motion and assessment of the severity of wall motion abnormality compared with MRI. The ability of LVO to improve scoring and inter-observer variability of regional wall motion at rest has important implications for stress echocardiography. Stress echocardiography is an established clinical tool with a high sensitivity and specificity for the diagnosis of coronary artery disease (CAD). During stress echo the diagnosis of CAD is based on detection of regional contractile dysfunction, and requires visualization of all myocardial segments to document or exclude abnormalities definitively. Reduced endocardial border definition is exacerbated during stress because of chest wall movement during hyperventilation and cardiac translational movement during tachycardia. With fundamental imaging, inadequate endocardial definition has been reported in up to 30% of stress echos [20]. In addition, Hoffman et al demonstrated that suboptimal studies have worse reproducibility and a poorer inter observer variability, with inter-institutional institutional observer agreement as low as 43% for studies with poor image quality[21] Tissue harmonic imaging, digital side by side analysis and standardised reporting criteria have alleviated but not overcome this problem[22]. Wall motion scoring and reproducibility during stress echo were even improved with air filled contrast agents and fundamental imaging[23]. Perflurocarbon filled agents demonstrated almost complete and consistent endocardial border definition [24], with superiority even to tissue harmonic imaging, and the greatest improvement being seen in patients with poorest image quality (26). Despite these clear advantages, the critical clinical question of whether LVO actually improves the accuracy of stress echo for diagnosis of CAD remains unanswered – Dolan [25] came closest by investigating 229 patients undergoing dobutamine echo followed by angiography, the largest study of contrast stress echo (for LVO) with angiography as the gold standard. As previously documented, the endocardial border definition and inter observer variability was superior with contrast. Their important finding was that there was comparable sensitivity, specificity and accuracy for the presence of coronary disease between patients with poor resting images using contrast LVO and those in whom standard imaging gave good resting images. Moreover, our recent work shows that the improvement of LVO is an important contributor to the incremental benefit of MCE (Figure 7 and additional files 10,11,12,13,14,15,16,17,18,19,20,21,22, and 23). Figure 7 End systolic frames of 4CV at rest (left) and post stress (right). Note there is no obvious difference in the shape of the cavity on the grey scale images. Importantly, the LVO images demonstrate a clear change in shape with the basal and mid lateral segments lagging, suggestive of LCx stenosis. In addition, the mid lateral segment has a perfusion defect which was not present at rest. Subtotal occlusion of the LCx was demonstrated at angiography. [See also additional files 10-23 for entire study]. Thus, left ventricular opacification by contrast echo: (1) improves the visualization of myocardial endocardial border definition; (2) improves the accuracy of ventricular volume assessment and estimation of ejection fraction compared with standard fundamental and harmonic imaging. (3) improves the ability to identify and grade resting wall motion abnormalities (4) provides superior endocardial visualization while imaging at peak dobutamine stress and can enable the accuracy of a dobutamine stress echo in a technically difficult patient to be at least as good as that of a patient with good resting images; (5) reduces the inter-observer variability for all of the above. From an economic standpoint, the use of contrast agents during stress echo has been calculated to be cost effective [26] with the cost of the contrast agent itself more than offset by savings incurred by reducing downstream repetitive testing, improved laboratory efficiency and a lower rate of false positive and negatives. However, the calculations are based on a formulas incorporating improved accuracy of the technique for the diagnosis of CAD, compared with standard imaging, which remains un-proven. In summary, the use of contrast for LVO is justified for standard or stress imaging of technically difficult patients, and possibly, for calculation of ventricular dimensions in patients whom accurate quantitative serial follow up is critical, eg. chemotherapy or valvular heart disease. Other clinical uses of contrast for left ventricular opacification include confirming or excluding the presence of left ventricular thrombus and delineating other left ventricular structures like pseudoaneurysm, apical hypertrophic cardiomyopathy (Figure 8 and additional files 24,25), and non-compacted left ventricles [27-29]. Figure 8 Use of contrast echo to identify a patient with apical HCM. Note the 'spade shaped LV cavity' on the contrast image [See also additional files 24 and 25] III. Myocardial contrast echocardiography The ischaemic cascade In the ischaemic cascade, hypoperfusion precedes wall motion abnormalities, which precede ECG changes and the onset of chest discomfort [30]. Stress echo, which provides an indirect marker of hypo-perfusion by recognition of wall motion abnormalities, is more than 80% sensitive and specific for detection of CAD [20]. The technique has two groups of limitations; 1) Interpretive. Wall motion analysis is fundamentally subjective [31], leading to the need for specific training and discordance between observers. There are particular problems in the assessment of the inferoposterior wall. 2) Dependence on the induction of ischaemia (increased myocardial oxygen demand) to provide diagnostic information. Many problems spring from this – dependence on performance of adequate stress, influence by beta blockade, limited sensitivity for mild (eg single vessel) CAD, poor capacity to identify the extent of CAD (eg. 50% sensitivity for the detection of multi vessel disease)[32]. SPECT perfusion imaging is the best-established clinical method to address perfusion directly and theoretically should be more sensitive than stress echo because hypoperfusion occurs earliest the ischaemic cascade. However, the difference between the methods is marginal because of the technical limitations of SPECT scanning, which also relies on subjective interpretation. If technically feasible there is clear role for echocardiography to assess myocardial perfusion. Ultrasound is more widely available, portable, avoids radiation and has a better spatial resolution than SPECT scanning (up to 1 mm, compared with >10 mm). An ideal technique would allow assessment of myocardial perfusion and wall motion simultaneously. Coronary circulatory physiology Understanding the pathophysiology of normal myocardial perfusion is essential for understanding detection of CAD with MCE. Contrast micro-bubbles act as pure intravascular tracers, traversing the myocardial vasculature, and contrasting with MRI tracers (which escape into the extravascular space) and radionuclides (which enter myocytes). Myocardial blood volume The coronary blood volume (CBV) encompasses the entire coronary system, which includes the epicardial arteries, the arterioles, the capillary network, venules, veins and the coronary sinus. Approximately one-third of the CBV resides within the ventricular myocardium. This myocardial blood volume (MBV) includes "microvessels" of < 300 microns in diameter, with approximately 90% of the total myocardial blood volume lying within the capillaries (6–7 microns in diameter)[33]. Assessment of myocardial perfusion with MCE involves processing backscattered signals received from micro-bubbles within the MBV, so that myocardial opacification almost entirely reflects backscatter from micro-bubbles flowing through the myocardial capillary compartment. There are 2 components of this myocardial blood flow appreciated with MCE i) the intensity of the backscatter signal from the micro-bubbles within the myocardium (brightness of its appearance on echo) – related to the myocardial blood volume, ii) the rate of increase in intensity after bubble destruction reflects the red blood cell velocity. Their product, represents myocardial blood flow. The response of MBF myocardial blood flow and it's components to stress is central to the application of perfusion imaging. Normal physiology of perfusion Because of the near complete extraction of O2 from red cells by the myocardium under basal conditions, any increase in myocardial O2 demand must rapidly translate into increases in coronary blood flow. In the absence of a coronary stenosis, when myocardial O2 demand is increased, there is sufficient vasodilator reserve to allow coronary flow to increase by a factor of 4–6 fold above resting levels. The work of Kaul, Wei and Lindner at the University of Virginia has shown that the ability to increase coronary flow in response to increased demand is achieved through a process of coronary autoregulation, controlled predominantly by the arterioles (See table 2). The coronary micro-circulation strives to maintain a minimum trans-capillary pressure of approximately 30 mmHg. In the absence of a coronary stenosis, a resting patient with a mean aortic pressure of 90 mmHg will have a pre-capillary pressure of ~45 mmHg. This natural resistance between the aorta and the capillaries (of ~45 mmHg) is provided by the arterioles[34] which present up to 60% of the total coronary vascular resistance. In the presence of increased myocardial oxygen demand, there is arteriolar vasodilation, reducing the resistance at the arteriolar level, which enables a higher precapillary pressure, translating into increased red blood cell velocity across the capillary network, and opening dormant capillary networks in order to maintain mean trans-capillary pressure, thus increasing the overall myocardial blood volume. Hence overall myocardial blood flow is increased. Importantly, pure vasodilator stress tends to increase red cell velocity without marked changes in overall myocardial blood volume. In the presence of a significant, non-critical (60–90%) epicardial artery stenosis at rest, completely normal resting blood flow is maintained by arteriolar vasodilation. As a result myocardial perfusion imaging techniques cannot detect defects at rest in patients with non-critical stenoses. Thus identification of perfusion defects in the setting of a non-critical coronary stenosis requires stress imaging to induce either ischaemia (increased myocardial oxygen demand) or hyperaemia (pharmacologically induced maximal arteriolar vasodilation). During stress we expect a 4–6 fold increase in flow to areas supplied by non-stenotic arteries, mediated by arteriolar vasodilation, increased red cell velocity and increased myocardial blood volume (opening of dormant capillary networks). Flow in the perfusion bed subtended by a significantly stenosed artery is not augmented, as resting arteriolar vasodilation is present, so limited augmentation of flow can be achieved with hyperaemia. In some cases the pre-capillary pressure drops significantly due to low distal coronary pressure and steal. With reduced pre-capillary pressure the only means of attempting to maintain normal trans-capillary pressure is to shut down capillary networks which were previously open (capillary de-recruitment). The net result is that direct comparison between regions subtended by a significant stenosis and normal territories reveals perfusion mismatch. In the presence of a critical stenosis, maximal arterial vasodilation maintains perfusion at rest. Any further reduction in distal coronary pressure directly translates into reduced precapillary pressure and reduced myocardial blood flow. Patients with critical lesions commonly have collateral vessels, making assessment of resting perfusion a complex phenomenon. Qualitative assessment of myocardial contrast for diagnosis of CAD There are three aspects to myocardial contrast opacification – signal intensity (equivalent to myocardial blood volume), pattern of filling and rate of filling. Signal intensity Myocardial blood volume is the easiest parameter to interpret qualitatively, and most studies have addressed differences in MBV between rest and stress in myocardial segments. Perfusion has traditionally been scored using a graded scale; 1 for homogenous perfusion, 0.5 for reduced perfusion and 0 for absent perfusion [8]. The presence of a stress induced perfusion defect not seen on the resting images indicates ischaemia, but in the absence of destruction-replenishment imaging, may appear normal in mild disease. The presence of a resting perfusion defect signifies infarction or artefact, with an infarction likely if there are associated regional wall motion abnormalities at rest, and if the defect conforms to the distribution of a coronary vascular territory. False positive defects often reflect technical limitations (Figure 9). Failure to obtain signal from a segment may be due to attenuation by overlying contrast, shadowing by rib or other structure, bubble destruction (especially in the apex) or failure to deliver sufficient contrast to enter the microcirculation. Figure 9 False positive defects with real-time MCE. Pseudo-apical defects are due to apical bubble destruction (a). Relocation of the focus from the base toward the apex (b) leads to "resolution" of the apical abnormality, but use of a mid-ventricular focus placement may lead to more problems with definition of the basal segments. This case also exemplifies attenuation of the basal lateral segment by contrast within the LV cavity. Perfusion distribution While most infarctions are shown as a transmural perfusion defect, stress-induced defects are often restricted to the subendocardium (Figure 10a,10b). The detection of this finding allows a greater level of confidence than does a transmural defect, or particularly epicardial defects (which may be artefactual). Figure 10 Contribution of regional shape changes to the identification of perfusion defects, including irregular wall contour in the apex (a) and mid-inferior segment (b), both associated with subendocardial defects. The C shape of the basal inferior segment complements the diagnosis of a perfusion defect in this segment. (c) The location of a defect has an important relationship to its likelihood of being a true or a false positive. Generally, defects on the left hand side of the image are most likely to be true positives and those restricted to the right side (anterior, lateral wall) should be considered very critically before they are identified as abnormal, as these are the most common sites of false positives. Fortunately, in our experience the apex is a more sensitive marker of LAD disease than the anterior wall, and is usually well seen. Likewise, a lateral segment should be matched with a posterior wall finding before circumflex disease is reported. The basal segments are also problematic – especially with low MI imaging – and in our experience, the basal inferior wall should be matched to an adjacent abnormal segment (basal septum, mid-inferior) to reduce false positive interpretations. The shape of the LV cavity is a clue to true positive findings. A "C-shaped" perfusion defect in the inferior wall (Figure 10c) is a common distribution with right coronary disease. Irregularities of the walls (Figure 10b) or apex (Figure 10a) or apical "beaking" (Figure 10c) may provide evidence of dyssynchrony, and are often associated with subendocardial perfusion abnormalities. Rate of filling The rate of replenishment after bubble destruction is dependent on coronary flow, and even if this is not quantified, rate of refill is a reliable and sensitive marker of a true positive defect. Using low MI techniques, micro-bubbles are infused until adequate LVO and myocardial opacification are achieved (Figure 11b). Several pulses of high mechanical index are delivered (Figure 11c), causing destruction of micro-bubbles in the beam elevation, such that the previously opacified myocardium is now empty (Figure 11d). The replenishment time of micro bubbles into the myocardium can be observed qualitatively (Figure 11e,11f,11g,11h). As the mean myocardial microbubble velocity is 1 mm/sec, and the beam elevation is approximately 5 mm, it would take up to 5 seconds (ie 5–6 heart beats at a resting heart rate from 60–80) for homogenous opacification of the myocardium at rest. With hyperaemia in the absence of stenosis, myocardial blood flow should increase 4–6 fold. Thus, images taken within 1–2 seconds following bubble destruction (ie 2–3) heart beats at a peak heart rate of 140) should demonstrate complete homogenous refill within this time frame, and failure to fill in a perfusion defect thus represents reduced myocardial blood flow. Perfusion defects noted on the post stress images, not evident on the resting images suggest ischaemia [35]. -see figures 12, 13 and 14 (and additional files 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, and 49) for evidence of LAD, RCA and multi-vessel CAD respectively. Assessment of end-systolic frames is preferable, despite the presence of more coronary flow in diastole, because the myocardium is thicker and there is less risk of contamination by the blood pool, and perhaps also because the capillary network is 'sealed' during systolic contraction with minimal flow in or out of the capillary compartment [36]. Figure 12 A clear apical defect is evident 2 beats post flash at peak stress (bottom line) which was not evident at rest (top line), consistent with LAD stenosis. [See additional files 26-39 for full case movies, additional file 40 for angiogram and additional file 49 for curve fits]. Figure 13 A basal and mid inferior defect is evident 2 beats post flash at peak stress which was not evident at rest, consistent with RCA stenosis. [See additional files 41-46 for movies and 47 and 48 for angiography]. Figure 14 On the 4CV, an apical and a subendocardial basal infero-septal defect are evident 2 beats post stress. The lateral wall is affected by artefact. On the 2CV, there is hypoperfusion of the inferior wall and subendocardial apical defect on the post stress images. This is consistent with multi-vessel disease. High MI techniques apply the same principles, with infusion until homogenous myocardial opacification. Imaging is then changed to a sequential intermittent mode, beginning with continuous high MI imaging (which results in bubble destruction and empties the myocardium). Images are then recorded intermittently, gated from the ECG (usually end systolic) with an incrementally lengthening triggering interval, ie 1:1 an image every cardiac cycle, then 1:2, and image every two cardiac cycles, then 1:3, 1:4 etc. The same principles are used for assessment of perfusion defects. A perfusion defect evident on 1:2 or 1:3 triggering at peak stress not seen at 1:5/6 triggering at rest is significant and reflects an ischaemic response to stress. Validation of qualitative contrast echo for diagnosis of CAD Qualitative assessment of perfusion with MCE was validated in open chest dogs using graded coronary stenosis [37]. The original landmark paper in humans [8] compared MCE (intermittent harmonic MCE with offline digital subtraction and colour coding) with sestamibi SPECT scanning in 30 patients undergoing dipyridamole stress to investigate known or suspected coronary disease. Normal segments showed a 91% concordance, and abnormal segments an 85% concordance, with 90% concordance by vascular territory. There was an overall 86% concordance for the detection of coronary disease. Subsequent studies have validated MCE following vasodilator or dobutamine stress using other imaging modalities, including power Doppler and real time power pulse inversion/power modulation in animals and in humans have produced similar results to SPECT [38-40]. A recent study using real time imaging was the first to report the use of MCE with exercise stress [41] (which is technically challenging because of cardiorespiratory movement and short duration of hyperaemia) and again demonstrated concordance between MCE and SPECT imaging. There was a 76% agreement between MCE and SPECT and an 88% agreement between the combination of wall motion and MCE with SPECT. This study incorporated the wall motion data from real time imaging, and despite the low frame rate, the results hint at the incremental benefit of combining the approaches. While numerous studies examining various stress and imaging modalities have demonstrated concordance between MCE and SPECT, there remains a paucity of data using quantitative coronary angiography (QCA) as the gold standard. The presence and extent of CAD by QCA has traditionally been used as the reference standard for the assessment of CAD, but few studies have compared MCE and angiography (Table 3) [42]. In 45 patients undergoing mainly dobutamine stress echocardiography, Cwajg et al showed real time contrast-enhanced imaging was more sensitive than standard stress echocardiography (87% vs 56%), and gave a better recognition of disease extent (85% vs 39%). These results were surprisingly unfavourable for standard imaging, perhaps reflecting the limitations of lower frame-rate for real-time imaging. Moreover, no specificity data were available from this study. In the study of exercise stress (41), only 44 of the 100 patients who had MCE and SPECT proceeded to angiography, and in this group, the sensitivity and specificity of wall motion and MCE were not significantly different. In a recent study of 85 prospectively recruited patients, the largest involving quantitative coronary angiography, we demonstrated the addition of contrast (for LVO and MCE), to standard ExE significantly enhanced the sensitivity of the test for detection of CAD from 74 to 91%, with a non-significant reduction in specificity (57). Table 3 Myocardial contrast echo studies with patients undergoing coronary angiography. Author CAD No CAD Sensitivity MCE Specificity MCE Frequency of angiography Cwajg [42] 32 13 87% _ All 45 Shimoni [41] 28 16 75% 100% 44 (44%) of 101 Heinle [10] 12 3 75% 67% 15 (12%) of 123 Wei [43] 15 - 100% - 15 (28%) of 54 Rocchi [55] 12 - 89% - 12 (48%) 25 Olszowska [56] 44 - 97% - All 44 Moir [57] 43 27 91% 70% 70 of 85 Quantitative myocardial contrast echo for diagnosis of CAD Like stress echo and nuclear perfusion imaging, MCE is also limited by the qualitative nature of the interpretation. Subtle differences in video intensity between vascular beds may not be visually evident, potentially reducing the sensitivity for the detection of stenosis. Quantitative methods may help to alleviate this limitation and possibly reduce intra-observer and inter-observer variability in assessment. The process of quantitative myocardial contrast echo was validated in open chest dogs using intermittent imaging, and the same destruction-replenishment approach is used for contemporary qualitative assessment [43]. At a steady state during continuous intravenous infusion of micro bubbles, the number of bubbles entering or leaving any capillary unit is constant and depends on the flow rate of the bubbles. If the micro bubbles are destroyed at time zero, the video intensity in a selected myocardial region is close to zero dB (black). With time, bubbles will replenish the beam elevation – the degree of replenishment into the beam elevation increases as the time after destruction is increased until eventually the entire ultrasound beam elevation is replenished and a plateau is reached, whereby no further increase in video-intensity/brightness can occur (Figures 15 and 16). Because this relationship was originally described with intermittent imaging, it is usually described in terms of video-intensity vs pulsing intervals. Figure 15 The raw signal intensity data from the apical septal segment is plotted against time after destruction. Figure 16 an exponential function curve is applied to allow calculation of A, beta and A*beta. The video intensity vs pulsing interval or time curve resembles an exponential growth function that can be fitted by the equation I = A (1-e-βt) where y is the video intensity at the pulsing interval or time t, A is the plateau video intensity and β represents the rate of rise of the slope. In this model the plateau myocardial video intensity (A) represents the myocardial blood volume. The rate of rise of the slope (β) represents the mean myocardial red blood cell velocity and their product (A*β) represents myocardial blood flow. Wei demonstrated an excellent linear relationship between absolute myocardial blood flow measured with radiolabelled microspheres and myocardial contrast echo derived myocardial blood flow. While ECG gated intermittent triggered harmonic imaging was used to validate these measurements, both power Doppler and real time techniques have subsequently demonstrated similar results in animal experiments ([44-46]). More recently, Wei evaluated the ability of MCE to calculate flow reserves from these measurements in humans ([47]), using intermittent imaging in 30 selected patients undergoing coronary angiography (11 of whom had no CAD and 19 had non-critical single vessel stenoses). Quantitative MCE and invasive measurement coronary flow (Doppler flow wire) were performed at rest and following vasodilator stress. In the normal subjects, myocardial blood flow velocity (β) and myocardial blood flow (A*β) reserves demonstrated a linear relationship to coronary blood flow reserve measured invasively. In patients with CAD, there were significant differences in MBF velocity reserves between patients with mild moderate and severe stenoses, and a MBF velocity reserve of < 1.8 indicated a >70% stenosis. Thus MBF reserve appears to be a feasible non-invasive measure of CBF reserve in humans, which may allow non-invasive assessment of CAD and micro-vascular dysfunction. More recently, Dawson investigated the use of quantitatively derived MBF reserves to diagnose CAD using SPECT as the gold standard. She demonstrated moderate feasibility, with quantitative MBF reserves from both high and low MI imaging able to identify perfusion defects ([48]). Low MI imaging had a lower sensitivity. Contrast Echo and myocardial viability in chronic CAD It is now well recognised that regional or global ventricular dysfunction does not necessarily imply irreversible necrosis. Hypokinetic, akinetic or dyskinetic, yet viable myocardium may be stunned or hibernating. Myocardial stunning occurs after a period of acute ischaemia, despite restoration of completely normal blood flow. The natural history of stunning is of spontaneous improvement in the viable myocardium over time. Hibernating myocardium is the term used to describe the presence of significant ventricular dysfunction in patients with chronic CAD, which recovers after revascularisation. Improvement in function of sufficient numbers of viable but hibernating segments is associated with symptomatic benefit and improved survival[49]. Sadly, many patients with chronic CAD and LV dysfunction have minimal viability, and revascularisation of these patients is associated with significant risk, minimal benefit and possibly worse outcome, hence the need for a reliable test for identification of viability. Radionuclide scanning, dobutamine echocardiography, MRI and PET scanning are currently available modalities, each with various advantages and disadvantages but similar efficacy for prediction of myocardial functional recovery after revascularization. MCE may also have an important role in this clinical setting. Viable myocardium is associated with preservation of the micro-vasculature, and as micro-bubbles act as pure intra-vascular tracers, the presence of myocardial perfusion by any MCE technique at rest implies viability (see figure 17). Using intra-coronary injection of bubbles, Nagueh demonstrated MCE was feasible and had similar accuracy to thallium SPECT and dobutamine echo for identification of functional recovery[50]. Using intravenous micro-bubble administration, MCE demonstrated comparable efficacy to SPECT and DSE. Importantly in both of these studies quantitative MCE was superior to qualitative assessment[51]. Unfortunately there remains a paucity of further data in this clinically important area. Figure 17 Resting apical 2-chamber view, 10 beats post-flash, demonstrating absent perfusion to the anterior myocardial wall. Safety of contrast echocardiography The incidence of reported adverse events in human trials (mainly investigating LV opacification) has been very low. There have been particular theoretical concerns raised about histologic abnormalities and cardiac marker elevation in animals with high MI imaging[52], and whilst serum tropinin levels are normal in humans after high MI imaging[53], recent work has demonstrated troponin I and myoglobin in coronary sinus samples of humans after high MI imaging[54]. In recent months the agent SonoVue (a phospho-lipid shell encapsulating sulphur hexafluoride gas), approved in Europe, was withdrawn from clinical use because of adverse events including fatalities related to idiosyncratic hypersensitivity reactions. At this stage, specific details about the dose of agent and imaging modality used in these cases is not available. IV. Conclusion Advances in micro-bubble development, combined with the development of contrast specific imaging modalities have enabled not only excellent LVO, but reliable qualitative and quantitative assessment of myocardial perfusion by ultrasound, following intravenous injections of micro-bubbles. Use of this technology during stress echo increases sensitivity and improves the non-invasive evaluation of CAD. Table 1 Technical details and clinical implications of contrast-specific imaging techniques High Power Intermittent Imaging Acquisition difficulty Amount of contrast used Amplitude of micro-bubble backscatter Dynamic range Greyscale or colour Processed Backscatter Myocardial perfusion Wall motion Artefacts Tissue Harmonic Imaging challenging low high wide grey harmonic yes no yes Ultraharmonic challenging low high wide grey harmonic yes no yes Pulse inversion challenging low high wide grey harmonic yes no yes++ Harmonic Power Doppler challenging low high wide colour harmonic yes no yes Low Power Continuous Imaging Power Pulse inversion easy high low narrow colour harmonic yes yes yes Power modulation easy high low narrow either fundamental yes yes yes Table 2 Relationship between stenosis severity and stress on capillary pressure, myocardial blood volume and red cell velocity. Arbitrary numbers given for illustrative purposes only. Distal coronary artery pressure Arteriolar resistance Pre-capillary pressure Trans-capillary pressure RBC velocity MBV No stenosis REST 90 mm Hg Normal 45 mmHg 45 mmHg 30 mmHg Normal Normal No stenosis STRESS 90 mm Hg ↓ Maximal 25 mmHg 65 mmHg 30 mmHg ↑ ↑ Stenosis REST 75 mm Hg ↓ Sub-maximal 30 mmHg 45 mmHg 30 mmHg Normal Normal Stenosis STRESS 60 mm Hg ↓ Maximal 25 mmHg 35 mmHg 30 mmHg ↓ ↓ Supplementary Material Additional File 1 Power Doppler 4CV at rest in normal subject. Click here for file Additional File 2 A4CV standard harmonic imaging Click here for file Additional File 3 Contrast LVO with standard harmonic imaging Click here for file Additional File 4 Real time MCE with LVO and myocardial perfusion. Click here for file Additional File 5 Contrast LVO with ideal machine settings to optimize image quality and frame rate Click here for file Additional File 6 3-D non contrast imaging Click here for file Additional File 7 3-D imaging with contrast Click here for file Additional File 8 Real time 3-D without contrast Click here for file Additional File 9 Real time 3-D with contrast. Click here for file Additional File 10 Mr CC PLAX view rest Click here for file Additional File 11 Mr CC PLAX view stress Click here for file Additional File 12 Mr CC PSSAX view rest Click here for file Additional File 13 Mr CC PSSAX view stress Click here for file Additional File 14 Mr CC A4CV rest Click here for file Additional File 15 Mr CC A4CV stress Click here for file Additional File 16 Mr CC A2CV rest Click here for file Additional File 17 Mr CC A2CV stress Click here for file Additional File 18 Mr CC ALAX view rest Click here for file Additional File 19 Mr CC ALAX view stress Click here for file Additional File 20 Mr CC contrast 4CV rest Click here for file Additional File 21 Mr CC contrast 4CV stress Click here for file Additional File 22 Mr CC contrast 2CV rest Click here for file Additional File 23 Mr CC contrast 2CV stress Click here for file Additional File 24 Apical HCM standard 4CV Click here for file Additional File 25 Apical HCM contrast rest Click here for file Additional File 27 Mr JD Rest PSAX Click here for file Additional File 28 Mr JD Rest A4CV Click here for file Additional File 26 Mr JD Rest PLAX Click here for file Additional File 29 Mr JD Rest A2CV Click here for file Additional File 30 Mr JD Rest ALAX view Click here for file Additional File 31 Mr JD Rest contrast 4CV Click here for file Additional File 32 Mr JD Rest contrast A2CV Click here for file Additional File 33 Mr JD Stress PLAX Click here for file Additional File 34 Mr JD Stress PSSAX Click here for file Additional File 35 Mr JD Stress A4CV Click here for file Additional File 36 Mr JD Stress A2CV Click here for file Additional File 37 Mr JD Stress ALAX view Click here for file Additional File 38 Mr JD Stress contrast 4CV Click here for file Additional File 39 Mr JD Stress contrast 2CV Click here for file Additional File 40 Mr JD angiogram. Click here for file Additional File 41 Mr T 4CV rest Click here for file Additional File 42 Mr T 4CV stress Click here for file Additional File 43 Mr T 2CV rest Click here for file Additional File 44 Mr T 2CV stress Click here for file Additional File 45 Mr T 2CV contrast rest Click here for file Additional File 46 Mr T 2CV contrast stress Click here for file Additional File 47 Mr T cath LCA Click here for file Additional File 48 Mr T cath RCA Click here for file Additional File 49 Mr JD curve fits A4CV Click here for file ==== Refs Reisner SA Ong LS Lichtenberg GS Myocardial perfusion imaging by contrast echocardiography with use of intracoronary sonicated albumin in humans J Am Coll Cardiol 1989 14 660 665 2768714 Vandenberg BF Feinstein SB Kieso RA Hunt M Kerber RE Myocardial risk area and peak gray level measurement by contrast echocardiography: effect of microbubble size and concentration, injection rate, and coronary vasodilation Am Heart J 1988 115 733 739 3354401 10.1016/0002-8703(88)90872-1 De Jong N Hoff L Ultrasound scattering properties of Albunex microspheres Ultrasonics 1993 31 175 181 8484195 10.1016/0041-624X(93)90004-J Wei K Skyba DM Firschke C Jayaweera AR Lindner JR Kaul S Interactions between microbubbles and ultrasound: in vitro and in vivo observations J Am Coll Cardiol 1997 29 1081 1088 9120163 10.1016/S0735-1097(97)00029-6 Burns PN Harmonic imaging with ultrasound contrast agents Clin Radiol 1996 51 50 55 8605774 Porter TR Xie F Kricsfeld D Armbruster RW Improved myocardial contrast with second harmonic transient ultrasound response imaging in humans using intravenous perfluorocarbon-exposed sonicated dextrose albumin J Am Coll Cardiol 1996 27 1497 1501 8626965 10.1016/0735-1097(96)00017-4 Halmann M Beyar R Rinkevich D Digital subtraction myocardial contrast echocardiography: design and application of a new analysis program for myocardial perfusion imaging J Am Soc Echocardiogr 1994 7 355 362 7917343 Kaul S Senior R Dittrich H Raval U Khattar R Lahiri A Detection of coronary artery disease with myocardial contrast echocardiography: comparison with 99mTc-sestamibi single-photon emission computed tomography Circulation 1997 96 785 792 9264483 Becher H Tiemann K Schlief R Luderitz B Nanda NC Harmonic Power Doppler Contrast Echocardiography: Preliminary Clinical Results Echocardiography 1997 14 637 11175004 Heinle SK Noblin J Goree-Best P Assessment of myocardial perfusion by harmonic power Doppler imaging at rest and during adenosine stress: comparison with (99m)Tc-sestamibi SPECT imaging Circulation 2000 102 55 60 10880415 Porter TR Xie F Silver M Kricsfeld D Oleary E Real-time perfusion imaging with low mechanical index pulse inversion Doppler imaging J Am Coll Cardiol 2001 37 748 753 11693747 10.1016/S0735-1097(00)01204-3 Dawson D Rinkevich D Belcik T Measurement of myocardial blood flow velocity reserve with myocardial contrast echocardiography in patients with suspected coronary artery disease: Comparison with quantitative gated Technetium 99m sestamibi single photon emission computed tomography J Am Soc Echocardiogr 2003 16 1171 1177 14608289 10.1067/S0894-7317(03)00646-1 Schiller NB Shah PM Crawford M Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms J Am Soc Echocardiogr 1989 2 358 367 2698218 Al Mansour HA Mulvagh SL Pumper GM Klarich KW Foley DA Usefulness of harmonic imaging for left ventricular opacification and endocardial border delineation by optison Am J Cardiol 2000 85 795 9 A10 12000067 10.1016/S0002-9149(99)00868-1 Grayburn PA Weiss JL Hack TC Phase III multicenter trial comparing the efficacy of 2% dodecafluoropentane emulsion (EchoGen) and sonicated 5% human albumin (Albunex) as ultrasound contrast agents in patients with suboptimal echocardiograms J Am Coll Cardiol 1998 32 230 236 9669275 10.1016/S0735-1097(98)00219-8 Schiller NB Shah PM Crawford M Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms J Am Soc Echocardiogr 1989 2 358 367 2698218 Hundley WG Kizilbash AM Afridi I Franco F Peshock RM Grayburn PA Administration of an intravenous perfluorocarbon contrast agent improves echocardiographic determination of left ventricular volumes and ejection fraction: comparison with cine magnetic resonance imaging J Am Coll Cardiol 1998 32 1426 1432 9809958 10.1016/S0735-1097(98)00409-4 Thomson HL Basmadjian AJ Rainbird AJ Contrast echocardiography improves the accuracy and reproducibility of left ventricular remodeling measurements: a prospective, randomly assigned, blinded study J Am Coll Cardiol 2001 38 867 875 11527647 10.1016/S0735-1097(01)01416-4 Hundley WG Kizilbash AM Afridi I Franco F Peshock RM Grayburn PA Effect of contrast enhancement on transthoracic echocardiographic assessment of left ventricular regional wall motion Am J Cardiol 1999 84 1365 1369 10614810 10.1016/S0002-9149(99)00577-9 Marwick TH Nemec JJ Pashkow FJ Stewart WJ Salcedo EE Accuracy and limitations of exercise echocardiography in a routine clinical setting J Am Coll Cardiol 1992 19 74 81 1729348 Hoffmann R Lethen H Marwick T Standardized guidelines for the interpretation of dobutamine echocardiography reduce interinstitutional variance in interpretation Am J Cardiol 1998 82 1520 1524 9874058 10.1016/S0002-9149(98)00697-3 Hoffmann R Marwick TH Poldermans D Refinements in stress echocardiographic techniques improve inter-institutional agreement in interpretation of dobutamine stress echocardiograms Eur Heart J 2002 23 821 829 12009723 10.1053/euhj.2001.2968 Porter TR Xie F Kricsfeld A Chiou A Dabestani A Improved endocardial border resolution during dobutamine stress echocardiography with intravenous sonicated dextrose albumin J Am Coll Cardiol 1994 23 1440 1443 8176104 Rainbird AJ Mulvagh SL Oh JK Contrast dobutamine stress echocardiography: clinical practice assessment in 300 consecutive patients J Am Soc Echocardiogr 2001 14 378 385 11337683 10.1067/mje.2001.111264 Dolan MS Riad K El Shafei A Effect of intravenous contrast for left ventricular opacification and border definition on sensitivity and specificity of dobutamine stress echocardiography compared with coronary angiography in technically difficult patients Am Heart J 2001 142 908 915 11685180 10.1067/mhj.2001.117608 Thanigaraj S Nease RF JrSchechtman KB Wade RL Loslo S Perez JE Use of contrast for image enhancement during stress echocardiography is cost-effective and reduces additional diagnostic testing Am J Cardiol 2001 87 1430 1432 11397374 10.1016/S0002-9149(01)01573-9 Stratton JR Lighty GW JrPearlman AS Ritchie JL Detection of left ventricular thrombus by two-dimensional echocardiography: sensitivity, specificity, and causes of uncertainty Circulation 1982 66 156 166 7083502 Moreno R Zamorano JL Almeria C Usefulness of contrast agents in the diagnosis of left ventricular pseudoaneurysm after acute myocardial infarction Eur J Echocardiogr 2002 3 111 116 12114095 10.1053/euje.2001.0130 Zhu H Muro T Hozumi T [Usefulness of left ventricular opacification with intravenous contrast echocardiography in patients with asymptomatic negative T waves on electrocardiography] J Cardiol 2002 40 259 265 12528647 Leong-Poi H Rim SJ Le DE Fisher NG Wei K Kaul S Perfusion versus function: the ischemic cascade in demand ischemia: implications of single-vessel versus multivessel stenosis Circulation 2002 105 987 992 11864930 10.1161/hc0802.104326 Hoffmann R Hanrath P Stress echocardiography: the scourge of subjective interpretation Eur Heart J 1995 16 1458 1459 8881833 O'Keefe JH JrBarnhart CS Bateman TM Comparison of stress echocardiography and stress myocardial perfusion scintigraphy for diagnosing coronary artery disease and assessing its severity Am J Cardiol 1995 75 25D 34D 7726110 10.1016/0002-9149(95)90108-6 Kaul S Jayaweera AR Coronary and myocardial blood volumes: noninvasive tools to assess the coronary microcirculation? Circulation 1997 96 719 724 9264473 Chilian WM Eastham CL Marcus ML Microvascular distribution of coronary vascular resistance in beating left ventricle Am J Physiol 1986 251 H779 H788 3766755 Lepper W Nowak B Franke A Myocardial contrast echocardiography, single-photon emission computed tomography, and regional function analysis for coronary stenosis description during vasodilator stress Am J Cardiol 2003 91 445 448 12586262 10.1016/S0002-9149(02)03243-5 Leong-Poi H Le E Rim SJ Sakuma T Kaul S Wei K Quantification of myocardial perfusion and determination of coronary stenosis severity during hyperemia using real-time myocardial contrast echocardiography J Am Soc Echocardiogr 2001 14 1173 1182 11734784 10.1067/mje.2001.115982 Firschke C Wei K Kaul S Quantification of the physiological relevance of a coronary stenosis using myocardial contrast echocardiography Coron Artery Dis 2000 11 203 209 10832553 10.1097/00019501-200005000-00002 Dawson D Rinkevich D Belcik T Measurement of myocardial blood flow velocity reserve with myocardial contrast echocardiography in patients with suspected coronary artery disease: Comparison with quantitative gated Technetium 99m sestamibi single photon emission computed tomography J Am Soc Echocardiogr 2003 16 1171 1177 14608289 10.1067/S0894-7317(03)00646-1 Spencer KT Grayburn PA Mor-Avi V Myocardial contrast echocardiography with power Doppler imaging Am J Cardiol 2000 86 479 481 10946053 10.1016/S0002-9149(00)00975-9 Wei K Crouse L Weiss J Comparison of usefulness of dipyridamole stress myocardial contrast echocardiography to technetium-99m sestamibi single-photon emission computed tomography for detection of coronary artery disease (PB127 Multicenter Phase 2 Trial results) Am J Cardiol 2003 91 1293 1298 12767419 10.1016/S0002-9149(03)00316-3 Shimoni S Zoghbi WA Xie F Real-time assessment of myocardial perfusion and wall motion during bicycle and treadmill exercise echocardiography: comparison with single photon emission computed tomography J Am Coll Cardiol 2001 37 741 747 11693746 10.1016/S0735-1097(00)01179-7 Cwajg J Xie F O'Leary E Kricsfeld D Dittrich H Porter TR Detection of angiographically significant coronary artery disease with accelerated intermittent imaging after intravenous administration of ultrasound contrast material Am Heart J 2000 139 675 683 10740151 10.1016/S0002-8703(00)90047-4 Wei K Jayaweera AR Firoozan S Linka A Skyba DM Kaul S Quantification of myocardial blood flow with ultrasound-induced destruction of microbubbles administered as a constant venous infusion Circulation 1998 97 473 483 9490243 Masugata H Cotter B Peters B Ohmori K Mizushige K DeMaria AN Assessment of coronary stenosis severity and transmural perfusion gradient by myocardial contrast echocardiography: comparison of gray-scale B-mode with power Doppler imaging Circulation 2000 102 1427 1433 10993863 Masugata H Lafitte S Peters B Strachan GM DeMaria AN Comparison of real-time and intermittent triggered myocardial contrast echocardiography for quantification of coronary stenosis severity and transmural perfusion gradient Circulation 2001 104 1550 1556 11571251 Leong-Poi H Le E Rim SJ Sakuma T Kaul S Wei K Quantification of myocardial perfusion and determination of coronary stenosis severity during hyperemia using real-time myocardial contrast echocardiography J Am Soc Echocardiogr 2001 14 1173 1182 11734784 10.1067/mje.2001.115982 Wei K Ragosta M Thorpe J Coggins M Moos S Kaul S Noninvasive quantification of coronary blood flow reserve in humans using myocardial contrast echocardiography Circulation 2001 103 2560 2565 11382724 Dawson D Rinkevich D Belcik T Measurement of myocardial blood flow velocity reserve with myocardial contrast echocardiography in patients with suspected coronary artery disease: Comparison with quantitative gated Technetium 99m sestamibi single photon emission computed tomography J Am Soc Echocardiogr 2003 16 1171 1177 14608289 10.1067/S0894-7317(03)00646-1 Allman KC Shaw LJ Hachamovitch R Udelson JE Myocardial viability testing and impact of revascularization on prognosis in patients with coronary artery disease and left ventricular dysfunction: a meta-analysis J Am Coll Cardiol 2002 39 1151 1158 11923039 10.1016/S0735-1097(02)01726-6 Nagueh SF Vaduganathan P Ali N Identification of hibernating myocardium: comparative accuracy of myocardial contrast echocardiography, rest-redistribution thallium-201 tomography and dobutamine echocardiography J Am Coll Cardiol 1997 29 985 993 9120185 10.1016/S0735-1097(97)00001-6 Shimoni S Frangogiannis NG Aggeli CJ Microvascular structural correlates of myocardial contrast echocardiography in patients with coronary artery disease and left ventricular dysfunction: implications for the assessment of myocardial hibernation Circulation 2002 106 950 956 12186799 10.1161/01.CIR.0000026395.19594.43 Chen S Kroll MH Shohet RV Frenkel P Mayer SA Grayburn PA Bioeffects of myocardial contrast microbubble destruction by echocardiography Echocardiography 2002 19 495 500 12356345 10.1046/j.1540-8175.2002.00495.x Borges AC Walde T Reibis RK Does contrast echocardiography with Optison induce myocardial necrosis in humans? J Am Soc Echocardiogr 2002 15 1080 1086 12373250 Vancraeynest D Hanet C Kefer J Does contrast echocardiography cause myocardial damage in patients? Circulation 2003 108 624 Rocchi G Fallani F Bracchetti G Non-invasive detection of coronary artery stenosis: a comparison among power-Doppler contrast echo, 99Tc-Sestamibi SPECT and echo wall-motion analysis Coron Artery Dis 2003 14 239 245 12702928 10.1097/00019501-200305000-00008 Olszowska M Kostkiewicz M Tracz W Przewlocki T Assessment of myocardial perfusion in patients with coronary artery disease. Comparison of myocardial contrast echocardiography and 99mTc MIBI single photon emission computed tomography Int J Cardiol 2003 90 49 55 12821219 10.1016/S0167-5273(02)00475-8 Moir S Haluska B Jenkins C Incremental Benefit of Myocardial Contrast to Combined Dipyridamole-Exercise Stress Echocardiography for the Assessment of Coronary Artery Disease Circulation 2004

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==== Front Health Qual Life OutcomesHealth and Quality of Life Outcomes1477-7525BioMed Central London 1477-7525-2-451534506210.1186/1477-7525-2-45ReviewThe Menopause Rating Scale (MRS) scale: A methodological review Heinemann Klaas [email protected] Alexander [email protected] Peter [email protected] Hermann PG [email protected] Frank [email protected] Lothar AJ [email protected] Do Minh [email protected] Center for Epidemiology & Health Research Berlin, Invalidenstr. 115, 10115 Berlin, Germany2 Berlex Canada Inc., 334 Avenue Avro, Pointe-Claire (Québec) H9R 5W5, Canada3 TNS Healthcare(Germany), Landsberger Str. 338, D-80687 Munich, Germany4 University Muenster, Dept. Obstetrics and Gynecology, Von-Esmarch-Strasse 56, 48149 Muenster, Germany5 Schering Deutschland GmbH, Max-Dorn-Str. 10, 10589 Berlin, Germany2004 2 9 2004 2 45 45 26 6 2004 2 9 2004 Copyright © 2004 Heinemann et al; licensee BioMed Central Ltd.2004Heinemann et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background This paper compiles data from different sources to get a first comprehensive picture of psychometric and other methodological characteristics of the Menopause Rating Scale (MRS) scale. The scale was designed and standardized as a self-administered scale to (a) to assess symptoms/complaints of aging women under different conditions, (b) to evaluate the severity of symptoms over time, and (c) to measure changes pre- and postmenopause replacement therapy. The scale became widespread used (available in 10 languages). Method A large multinational survey (9 countries in 4 continents) from 2001/ 2002 is the basis for in depth analyses on reliability and validity of the MRS. Additional small convenience samples were used to get first impressions about test-retest reliability. The data were centrally analyzed. Data from a postmarketing HRT study were used to estimate discriminative validity. Results Reliability measures (consistency and test-retest stability) were found to be good across countries, although the sample size for test-retest reliability was small. Validity: The internal structure of the MRS across countries was astonishingly similar to conclude that the scale really measures the same phenomenon in symptomatic women. The sub-scores and total score correlations were high (0.7–0.9) but lower among the sub-scales (0.5–0.7). This however suggests that the subscales are not fully independent. Norm values from different populations were presented showing that a direct comparison between Europe and North America is possible, but caution recommended with comparisons of data from Latin America and Indonesia. But this will not affect intra-individual comparisons within clinical trials. The comparison with the Kupperman Index showed sufficiently good correlations, illustrating an adept criterion-oriented validity. The same is true for the comparison with the generic quality-of-life scale SF-36 where also a sufficiently close association has been shown. Conclusion The currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging women in different regions and over time, it suggests a high reliability and high validity as far as the process of construct validation could be completed yet. MRSHealth Related Quality of LifeQuestionnairesReliabilityValidity ==== Body Background The interest of clinical research in aging women and males increased in recent years and thereby the interest to measure health-related quality of life and symptoms. Women, as do men, experience an age-related decline of physical and mental capacity. They observe symptoms such as periodic sweating or hot flushes, impaired memory, lack of concentration, nervousness, depression, insomnia, and bone – joint complaints. The Menopause Rating Scale (MRS) is a health-related quality of life scale (HRQoL) and was developed in response to the lack of standardized scales to measure the severity of aging-symptoms and their impact on the HRQoL in the early 1990s. Actually, the first version of the MRS was to be filled out by the treating physician but methodological critics lead to a new scale which can easily be completed by women, not by their physician [1,2]. The validation of the MRS started some years ago [2-6] aiming at establishing an instrument to measure HRQoL that can easily be completed. The aims of the MRS were (1) to enable comparisons of the symptoms of aging between groups of women under different conditions, (2) to compare severity of symptoms over time, and (3) to measure changes pre- and post-treatment [4-6]. The MRS was formally standardized according to psychometric rules and initially published in German [2]. During the standardization of this instrument, three independent dimensions were identified explaining 59% of the total variance (factor analysis): psychological, somato-vegetative, and urogenital sub-scale. The MRS consists of a list of 11 items (symptoms or complaints). Each of the eleven symptoms contained in the scale can get 0 (no complaints) or up to 4 scoring points (severe symptoms) depending on the severity of the complaints perceived by the women completing the scale (an appropriate box is to be ticked). The scoring scheme is simple, i.e. the score increases point by point with increasing severity of subjectively perceived symptoms in each of the 11 items (severity 0 [no complaints]...4 scoring points [very severe symptoms]). The respondent provides her personal perception by checking one of 5 possible boxes of "severity" for each of the items. This can be seen in the questionnaires in the additional files linked to this publication. The composite scores for each of the dimensions (sub-scales) is based on adding up the scores of each item of the respective dimensions. The composite score (total score) is the sum of the dimension scores. The three dimensions, their corresponding questions and the evaluation are detailed and summarized in an attached file linked to this publication [see Additional file 1]. The MRS scale became internationally well accepted. The first translation was into English [7]. Other translations followed [8], i.e. taking international methodological recommendations [9,10] into consideration. Currently, the following versions are available: Brazilian, English, French, German, Indonesian, Italian, Mexican/Argentine, Spanish, Swedish, and Turkish language. These versions are available in a published form, and can be downloaded in PDF-format from the internet (see reference 8 and ). Like in other QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity, and this in addition to the conventional psychometric requirements of test reliability and validity. The aim of this paper is to present additional psychometric data to discuss the methodologically relevant characteristics of the MRS scale. Methods The development of the scale, instrument characteristics (item selection, scaling), and norms and standardized scores have been published elsewhere [2-5]. This applies also for a few data that have been published on test-retest stability and criterion-dependent validity [3,6]. During the last two years a number of smaller and larger investigations were made from different groups to further check methodological features of the scale. We performed recently a large, multinational survey to represent the situation across nine countries and cultures using existing and for the respective countries representative panels between November 2001 and February 2002 to get information about knowledge, attitudes and behaviour related to hormonal treatment in women aged 40–70 years: Europe (Germany, France, Spain, Sweden), North America (USA), Latin America (Mexico, Argentine, Brazil), and as example for Asia – Indonesia. Study participants were accrued as a random sample of females aged 40 to 70 years from existing population panels. The sample size in each of the countries was about 1000 females aged 40–70 years, with exception of USA (n = 1500). The participation rates ranged between 46 and 94% across countries. The demographic details of the sample are: On average, about tertiles of the respondents were under 50 years, between 50–59, and over 60 years old in most of the countries, however, about 50% were less than 50 years in Indonesia and in Brazil. The majority of respondents reported a Christian religion in Europe (range: 74% (Germany) to 96% (Spain), 85% in USA, and in Latin America (range: 95% (Argentina) to 97% (Mexico). The use of the MRS was part of this survey, i.e. multinational data became available to reconsider methodological issues more thoroughly such as internal structure of the scale, reliability (internal consistency alpha), and reference values for different population. For the purposes of reliability assessment we performed a few preliminary studies with a test-retest approach. These small, descriptive studies of community samples of women aged 40–70 were done in summer and fall 2002 by local collaborators in the respective countries, but they were done separately and independent from the main study. These studies were done just for orientation with convenience samples – not representative for the respective population. There is only one intervention study (before and after hormonal treatment) available to our knowledge. This study has been published [6] but not with regard to methodologically relevant results of the MRS. These data will be published soon. With these data available, we were able to scrutinize many methodological characteristics of the MRS scale to review most fundamental psychometric characteristics as well validity parameters. Results and Discussion Reliability The assessment of scientific measurements depends first of all on the evidence of replicability (consistency) and test-retest reliability. In contrast to systematic and random variation, reliability gives an estimate of method-related measurement error which should be low not to hide or dilute intended systematic changes – due to treatment for example. Table 1 show the internal consistency measured with Cronbach's Alpha. The consistency coefficients range between 0.6 and 0.9 across countries for the total score as well the scores in the three domains. This is indicative for a very acceptable consistency of the MRS scale in our opinion. Moreover, there is no evidence that the scale works different in so many different countries in four continents. Table 1 Internal consistency coefficients (alpha) for the MRS scale across countries: total score and scores for the psychological, somatic, and urogenital domain. Data from the Nine-Country Study International Europe North America Latin America Asia Total Overall Sweden Germany France Spain USA Overall Mexico Brazil Argentina Indonesia N 9907 4465 1490 1050 941 984 1440 3002 1002 1000 1000 1000 Total score 0.83 0.86 0.85 0.84 0.86 0.86 0.88 0.86 0.87 0.86 0.83 0.84 Psychologic ascore 0.87 0.88 0.88 0.86 0.89 0.86 0.90 0.85 0.86 0.87 0.81 0.79 Somatic score 0.66 0.64 0.65 0.64 0.64 0.61 0.70 0.66 0.65 0.69 0.64 0.69 Urogenital score 0.65 0.65 0.64 0.63 0.67 0.67 0.70 0.60 0.62 0.55 0.62 0.65 The test-retest correlation coefficients (Pearson's correlation) support the suggestion of a good temporal stability of the total scale and its three sub-scales (Table 2), although most of the assessments across countries are based on very small numbers and convenience samples not claiming to be representative for the respective population. The intention of these pilot studies was to get a preliminary idea about retest stability. Larger sample sizes are required to permit final conclusions for individual countries / languages. Table 2 Test-retest coefficients (Pearson's correlation coefficient "r") for the MRS scale across countries: total score and scores for the psychological, somatic, and sexual sub-scale. All Europe Latin America Asia Overall Overall Germany UK France Spain Portugal Sweden Turkey Overall Argentina Brazil Overall Indonesia N 349 259 73 30 36 30 30 30 30 60 30 30 30 30 Total score 0,90 0,92 0,82 0,80 0,89 0,93 0,96 0,90 0,90 0,81 0,78 0,82 0,84 0,84 Psychological score 0,84 0,87 0,76 0,72 0,88 0,92 0,91 0,79 0,82 0,72 0,66 0,76 0,71 0,71 Somatic score 0,89 0,90 0,80 0,85 0,82 0,88 0,97 0,95 0,93 0,85 0,86 0,85 0,81 0,81 Urogen. score 0,86 0,89 0,77 0,82 0,94 0,98 0,95 0,87 0,89 0,73 0,67 0,74 0,50 0,50 The test-retest coefficients of the total score range between 0.8 and 0.96 across Europe, North and Latin America, and Asia. When it comes to the subscales with much fewer items, the variation increased and some of the coefficients went down to 0.5 (urogenital domain in Indonesia). Altogether, the test-retest stability over a time period of two weeks aggregated at the international level supports the notion of a very acceptable test-retest reliability of the total scale and their three sub-scales. Although there is an impressive set of information currently available concerning the reliability of the MRS scale, there are also limitations: Small sample sizes prevent a final conclusion regarding test-retest reliability in some of the languages the scale has been translated in. Validity Similar to reliability which assesses the consistency of measurement, the validity estimates if a scale measures what it intends to measure. But whereas reliability can be determined straight forward with very few indicators, the validity is almost always a continuous process (construct validation). It is a process of accumulating evidence for a valid measurement of what is purposed. Therefore, the currently available data are already fairly comprehensive and do pave the way for a focussed and continuing validation process. Internal structure of the MRS across countries The first step of validation is usually to multivariately demonstrate a similar internal structure ("dimensions") of a given scale through factor analysis. The first factorial analysis in 1996 was applied to identify the dimensions of the scale. Three dimensions of symptoms/complaints were identified [2]: a psychological, a somato-vegetative, and a urogenital factor that explained 58.8% of the total variance. The recent large, multinational survey in nine countries of four continents provided data to compare with the initial standardisation sample of the MRS. The question was: Is the internal structure of the MRS results comparable among different countries or cultures. Astonishingly similar factor loadings of the 11 items of the 3 domains of the MRS were observed (Table 3). The same applies for the individual countries of the respective regions (data not shown). Although the prevalence of menopausal symptoms may slightly differ among regions/cultures (see later), the structure of complaints/symptoms seems to be pretty much the same. It suggests that the scale measures constantly the same phenomenon which speaks in favour of the translation/cultural adaptation of the scale. Table 3 Internal structure of the MRS scale across countries in 9 countries of four continents (2002) compared with the initial analysis of a German sample in 1996. Principal component analysis, Varimax rotation. Complaints (item number in MRS), numbers, and country groups. Only factor loadings ≥ 0.5 are shown. Complaints (item number) N Flushes(1) Heart(2) Sleep(3) Joints(11) Depress(4) Irritabil(5) Anxiety(6) Exhaust(7) Sexual(8) Bladder(9) Dryness(10) Germany, 1996 479 somatic 0.8 0.7 0.6 0.5 psychologic 0.8 0.7 0.8 0.6 urogenital 0.7 0.8 0.8 All countries, 2002 10297 somatic 0.7 0.8 0.5 0.5 psychologic 0.8 0.8 0.8 0.7 urogenital 0.7 0.6 0.8 Europe, 2002 4791 somatic 0.7 0.7 0.6 0.6 psychologic 0.8 0.9 0.8 0.6 urogenital 0.7 0.6 0.8 N.-America (USA) 1500 somatic 0.7 0.7 0.7 0.5 psychologic 0.8 0.9 0.8 0.6 urogenital 0.5 0.8 0.6 0.8 Latin America, 2002 3006 somatic 0.5 0.9 0.5 0.4 psychologic 0.5 0.8 0.8 0.8 0.7 urogenital 0.6 0.7 0.8 Asia (Indonesia) 1000 somatic 0.9 0.6 0.5 0.5 psychologic 0.8 0.8 0.8 0.5 urogenital 0.8 0.5 0.9 However, there are indications that the domains could be somewhat overlapping and not as entirely independent as the statistical model suggests: Muscle or joint problems got a loading of 0.5 in the somatic and urogenital domain (USA), and sleep disturbances both 0.5 in somatic and psychological domain (Latin America). These two items had similar problems in Spain, Mexico, and Brazil but not in other countries (data not presented in table 3). In clinical studies intra-individual comparisons over time (before/after treatment) will be the main criterion which might not be affected by potential slight differences in the patient reported outcome structure. Therefore the general agreement in the internal structure of the MRS scale across country groups, even accepting the possibility of slight differences in two items (cf. Table 3), suggests that the scale can very well be used in clinical studies – even including different countries. Sub-scores and total score correlations The relations among the sub-scales and the aggregate total scale are patterns that are important in the methodological assessment of a scale. In an ideal world, the correlations between subscales (supposed to be independent due to the statistical model) would be closer to 0 than the correlations with the construct of the aggregate total score to which all sub-scales should significantly contribute. But that is theory; Table 4 shows only somewhat lower correlations among sub-scales (0.4–0.7) as compared with correlation of sub-scales with the total score (0.7–0.9). This is less different than one would have wished. It suggests that the sub-scales are not as independent from each other as one would expect them to be – based on a factorial analysis with orthogonal factors. The situation was similar in the four regions listed in Table 4 and in the individual countries belonging to these regions. It is important to realize how similar these correlation coefficients are among countries/aggregates. This is suggestive of pretty similar features of the MRS scale across the countries of this review. Table 4 Domain score – total score correlations of the MRS scale across four country groups. Community samples. Domains Psychological score Somatic score Urogenital score Europe (n = 4246) Total score 0.9 0.9 0.7 Psychological score -- 0.6 0.5 Somatic score -- -- 0.5 North America (n = 1376) Total score 0.9 0.9 0.8 Psychological score -- 0.7 0.5 Somatic score -- -- 0.5 Latin America (n = 3001) Total score 0.9 0.9 0.7 Psychological score -- 0.7 0.5 Somatic score -- -- 0.5 Asia (n = 1000) Total score 0.9 0.9 0.7 Psychological score -- 0.7 0.4 Somatic score -- -- 0.4 Compatibility of MRS reference values for different population There are different categories of severity of complaints or problems with QoL. For the comparison of these categories across countries or cultures it is important to understand the prevalence of complaints. Currently, there is only one table with reference values and definitions published – for the German population from the initial standardization of the MRS [2]. Are these reference values applicable for other countries/cultures? The data of our large multicultural survey permitted such comparisons. The mean values (SD) of the MRS total score and the three domains are depicted in Table 5. The mean values of the total score and the 3 domain scores are not statistically significantly different between Europe and North America. Thus, there is no evidence yet to exclude direct comparisons of MRS values between these regions. Table 5 Mean values and standard deviation of MRS total score and 3 domains. Results from a large, multinational survey (see methods) Total score Psychological Score Somato-vegetative Score Urogenital Score n Mean (SD) n Mean (SD) n Mean (SD) n Mean (SD) Europe 4246 8.8 (7.1) 4453 3.4 (3.4) 4465 3.6 (2.9) 4465 1.9 (2.2) N.-America (USA) 1376 9.1 (7.6) 1426 3.4 (3.5) 1440 3.8 (3.1) 1437 2.0 (2.3) Lat.-America 3001 10.4 (8.8) 3002 4.9 (4.5) 3006 4.1 (3.6) 3005 1.4 (2.2) Asia 1000 7.2 (6.0) 1000 2.9 (2.9) 1000 3.3 (2.7) 1000 1.0 (1.6) However, the total, psychological and somatic scores were systematically higher in Latin America, and systematically (significantly) lower in Asia (Indonesia) than in Europe/North America. The urogenital scores were significantly lower in Latin America/Indonesia than in Europe/US. Obviously the perception of the prevalent symptoms depends on cultural factors – or the symptoms show real differences in prevalence. Thus, direct comparisons of MRS scores between Europe/North America on the one side and regions in Latin America and Asia cannot are not recommended. This does not effect intra-individual comparisons (e.g., pre/ post therapy) within these countries and it may also very little affect the comparison of relative changes (pre/post treatment) among different countries. The latter is a hypothesis and needs further evidence form research/experience. Similar mean values could still have a different distribution across the proposed four categories of "severity of complaints": no/little symptoms, mild, moderate, and severe complaints, i.e. for the total scale and the three domains. The prevalence of these categories across the four regions studied is seen in Table 6. The comparison of the prevalence (and 95% confidence interval) showed that the above discussed differences between Europe/US and Latin-America or Indonesia very much depend on the severity of complaints. Whereas the differences in the psychological domain were less impressive, the dissimilarity was most pronounced in the urogenital domain and less also in the somatic domain. Whether this is due to different perception of identical symptoms (differences in the appearance of symptoms or both) remains a speculation. This however needs to be considered when direct comparisons among different cultures are intended. The prevalence of different "degrees of severity" of menopausal symptoms measured with the MRS was found to be almost identical in the aggregate of Europe and North America. Table 6 Comparison of "degree of severity" of the MRS and its domains. Prevalence in percent (%) and 95% confidence interval (in parenthesis) in the population sample studied in the respective regions (see methods) Europe North America Latin America Asia Total score No, little (0–4) 28.8 (+/-1.3) 28.0 (+/-2.3) 31.0 (+/-1.7) 40.2 (+/-3.0) Mild (5–8) 21.9 (+/- 1.2) 23.9 (+/-2.2) 20.2 (+/-1.4) 27.5 (+/-2.8) Moderate (9–16) 25.1 (+/-1.2) 25.7 (+/-2.2) 26.2 (+/-1.6) 22.8 (+/-2.6) Severe (17+) 24.3 (+/- 1.2) 22.5 (+/-2.1) 22.7 (+/-1.5) 9.5 (+/-1.8) Psychological domain No, little (0–1) 35.4 (+/-1.4) 36.8 (+/-2.4) 36.8 (+/-1.6) 41.3 (+/-3.1) Mild (2–3) 21.8 (+/-1.2) 21.9 (+/-2.1) 21.9 (+/-1.4) 25.4 (+/-2.7) Moderate (4–6) 19.5 (+/-1.1) 18.7 (+/-2.0) 18.7 (+/-1.4) 21.3 (+/-2.6) Severe (7+) 23.4 (+/-1.2) 22.5 (+/-2.1) 22.5 (+/-1.7) 12.0 (+/-2.0) Somato-vegetative domain No, little (0–2) 39.5 (+/-1.4) 37.9 (+/-2.4) 42.1 (+/-1.8) 46.8 (+/-3.1) Mild (3–4) 22.6 (+/-1.2) 25.6 (+/-2.2) 19.4 (+/-1.4) 27.0 (+/-2.8) Moderate (5–8) 24.2 (+/-1.2) 24.3 (+/-2.2) 25.6 (+/-1.6) 20.8 (+/-2.5) Severe (9+) 13.7 (+/-1.0) 12.1 (+/-1.7) 12.9 (+/-1.2) 5.4 (+/-1.4) Urogenital domain No, little (0) 34.3 (+/-1.3) 33.4 (+/-2.4) 28.2 (+/-1.8) 55.6 (+/-3.1) Mild (1) 17.2 (+/-1.1) 17.0 (+/-2.0) 18.6 (+/-1.1) 18.6 (+/-2.4) Moderate (2–3) 23.0 (+/- 1.2) 24.2 (+/-2.2) 21.8 (+/-1.3) 17.0 (+/-2.3) Severe (4+) 25.6 (+/-1.2) 25.4 (+/-2.2) 31.4 (+/-1.3) 8.8 (+/-1.8) Criterion-oriented validity: correlation with other scales In fact, the comparison with other scales of similar purpose is important. It is known from other quality of life scales that comparisons with scales with similar purposes are much more important than comparisons with so-called objective parameters such as exercise tests, physiological or chemical parameters – in our case with hormones. Health related quality of life should be validated against quality of life measured with other generic QoL scales (e.g., SF-36), and against specific instruments to measure symptoms in aging women (e.g. Kupperman index). These data were published elsewhere [6,11] but will be briefly summarized in the context of this review. Kupperman Index Although the Kupperman Index was not validated according to psychometric standards it is still in use in the medical practice to monitor menopausal symptoms. Therefore a comparison with the fully standardized MRS seems to be reasonable. If one divides the distribution of both scales into quartiles and compares the frequencies, both instruments were found to be closely associated: Kendall's tau-b coefficient 0.75 (95% CI 0.71–0.80) [6]. Similar was the Pearson correlation coefficient with r= 0.91(95% CI 0.89–0.93). The two scales can be regarded as measuring the same phenomena. However, some methodological problems of the Kupperman Index were identified in this comparison (see [6] for details). Generic QoL Scale SF-36 Two sub-scales of the multi-domain quality of life scale SF-36 was compared with the MRS: the somatic sum score (with somatic domain of MRS) and the psychologic sub-scales of both instruments. Both somatic domains were sufficiently well and significantly associated: Kendall's tau-b = 0.43 (95% CI 0.52–0.35); Pearson correlation coefficient r= 0.48 (95% CI 0.58–0.37). That means, the higher the score in the somatic dimension of the MRS, the lower the quality of life according to the somatic sum-score of the SF-36 [6,11]. Similar was the results of the comparison of the psychological scores of both instruments: Kendall's tau-b = 0.49 (95% CI 0.56–0.41); Pearson correlation coefficient r= 0.73 (95% CI 0.81–0.65). Discriminative validity i.e., the ability of the scale to accurately measure treatment effects and to predict the clinically based assessment of physicians, was not analysed so far. At present, there is one post-marketing study that can be used to preliminary assess discriminative validity. The results will be published soon elsewhere. To this end, many clinicians understand the term "validity" and mean high utility for clinical work or research. Conclusions The MRS scale is a standardized HRQoL scale with good psychometric characteristics. The use in many countries offered the possibility to compare the test characteristics across countries. Reliability measures (consistency and test-retest stability) were found to be good in all countries where data were obtained – however, some samples were very small and therefore considered as preliminary information. The validity was measured in its various forms: The internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon in women with complaints. The sub-sores and total score correlations showed high coefficients with the total score and less among the sub-scales. This however indicates that the subscales are not fully independent in practice. Comparisons of reference values from different populations showed that the MRS scores can easily be compared between Europe and North America/US. Direct comparisons between Europe/North America and Latin American countries and Asia (Indonesia) should be considered with caution because the severity of reported symptoms seems to differ. The reasons are not clear, further research is needed. The comparison with other scales for menopausal symptoms (Kupperman Index) showed a sufficiently close association and correlation coefficients, i.e. illustrating a good criterion-oriented validity. The same is true for the comparison with the generic QoL scale SF-36 where also high correlation coefficients have been shown. Thus, the currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging males over time or intervention. It suggests a high reliability and high validity as far as the process of construct validation could be completed. Authors' contributions KH: responsible for drafting the manuscript and running analyses. AR: responsible for designing and overseeing the multinational survey (2001/2002), contributed to writing and revising of the paper. PP: co-ordination of the field work of the multinational survey, setting up the initial database, and contributed to writing of the paper, responsibility in developing/validating the MRS scale. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript. FS: Provided data of a clinical study, contributed to writing of the manuscript. LAJH: responsible for the collection and evaluation of the data, and involved in writing/revising the paper. DMT: responsible for checking the integrated database, responsible for several analyses regarding validity, and contributed to writing of the paper. Acknowledgements The large multinational survey was sponsored by Schering AG Berlin; the field work done by teams of NFO Worldwide (now TNS Worldwide), TNS Healthcare Munich, co-ordinated by ZEG Berlin; we thank the respective central and local teams. We are grateful to having received a part of the database for our methodological evaluation of the MRS scale. 1We thank the marketing research institute IM Leipzig in Leipzig (Germany) for coordinating the small test-retest investigation in several countries to get a first orientation about the magnitude of the test-retest correlations. ==== Refs Hauser GA Huber IC Keller PJ Lauritzen C Schneider HPG Evaluation der klinischen Beschwerden (Menopause Rating Scale) Zentralbl Gynakol 1994 116 16 23 8147175 Potthoff P Heinemann LAJ Schneider HPG Rosemeier HP Hauser GA Menopause-Rating Skala (MRS): Methodische Standardisierung in der deutschen Bevölkerung Zentralbl Gynakol 2000 122 280 286 10857215 Heinemann K Assmann A Möhner S Schneider HPG Heinemann LAJ Reliabilität der Menopause-Rating-Skala (MRS). Untersuchung für die Deutsche Bevölkerung Zentralbl Gynakol 2002 124 161 163 12070795 10.1055/s-2002-32268 Schneider HPG Heinemann LAJ Rosemeier HP Potthoff P Behre HM The Menopause Rating Scale (MRS): Reliability of scores of menopausal complaints Climacteric 2000 3 59 64 11910611 Schneider HPG Heinemann LAJ Rosemeier HP Potthoff P Behre HM The Menopause Rating Scale (MRS): Comparison with Kupperman Index and Quality of Life Scale SF-36 Climacteric 2000 3 50 58 11910610 Schneider HPG Rosemeier HP Schnitker J Gerbsch S Turck R Application and factor analysis of the menopause rating scale [MRS] in a post-marketing surveillance study of Climen® Maturitas 2000 37 113 124 11137330 10.1016/S0378-5122(00)00177-8 Schneider HPG Heinemann LAJ Thiele K The Menopause Rating Scale (MRS): Cultural and linguistic translation into English Life and Medical Science Online 2002 3 DOI:101072/LO0305326 Heinemann LAJ Potthoff P Schneider HPG International versions of the Menopause Rating Scale (MRS) Health Qual Life Outcomes 2003 1 28 12914663 10.1186/1477-7525-1-28 Anonymous Trust introduces new translation criteria Medical Outcomes Trust Bulletin 1997 5 2 4 Acquadro C Jambon B Ellis D Marquis P Spilker B Languages and translation issues Quality of Life and Pharmaco-Econonomics in Clinical Trials 1996 2 575 585 Schneider HPG Behre HM Schneider HPG Contemporary evaluation of climacteric complaints: Its impact on quality of life Hormone replacement therapy and quality of life. The Parthenon Publishing Group 2002 Boca Raton, London, New York, Washington 45 61 Greene JG Schneider HPG Measuring the symptom dimension of quality of life: General and menopause-specific scales and their subscale structure Hormone replacement therapy and quality of life. The Parthenon Publishing Group 2002 Boca Raton, London, New York, Washington 35 43

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Health Qual Life Outcomes. 2004 Sep 2; 2:45

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Health Qual Life Outcomes

10.1186/1477-7525-2-45

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==== Front World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central London 1477-7819-2-291533933210.1186/1477-7819-2-29Case ReportRecurrence of primary extramedullary plasmacytoma in breast both simulating primary breast carcinoma Kaviani Ahmad [email protected] Mansoor [email protected] Maryam [email protected] Sedigheh [email protected] Department of Surgery, Tehran University of Medical Sciences, Tehran, Iran2 Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran3 Iranian Center for Breast Cancer, Tehran, Iran4 Professor of Pathology, Department of Pathology, Ohio State University, Ohio, USA5 Director, Iranian Center for Breast Cancer, Tehran, Iran2004 31 8 2004 2 29 29 28 4 2004 31 8 2004 Copyright © 2004 Kaviani et al; licensee BioMed Central Ltd.2004Kaviani et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Extramedullary myelomas (plasmacytoma) are malignant proliferations of plasma cells in the absence of bone involvement. When they occur in the soft tissue they usually involve the upper respiratory tract and oral cavity. Extramedullary plasmacytomas of breast are uncommon. Case presentation A 70 year-old woman with bilateral breast masses underwent excisional biopsy for suspected primary carcinoma that subsequently proved to be a recurrence from extramedullary plasmacytoma of the mediastinum. This was diagnosed and treated 5-years prior to appearance of breast lumps. Conclusion Though uncommon, considering the possibility of metastatic carcinoma and primary, secondary or recurrent lymphoproliferative disease presenting as a breast mass may avoid unnecessary surgeries. ==== Body Background Extramedullary myelomas (plasmacytoma) are malignant proliferations of plasma cells in the absence of bone involvement. When occur in the soft tissue, it usually involve the upper respiratory tract and oral cavity [1]. Plasmacytomas of breast are very rare. This report describes a patient with bilateral breast masses who underwent excision biopsy for suspected primary carcinoma that subsequently proved to be a recurrence from extramedullary plasmacytoma of mediastinum treated 5 years ago. To the best of our knowledge, this is the first case report of bilateral recurrence of a primary extramedullary plasmacytoma in breast tissues after a long disease-free interval. Case presentation A 70 year-old woman with a one-month history of bilateral breast masses was referred to our cancer center for surgical evaluation. There was no associated breast pain, skin change or nipple discharge. There was no history of bone pain, weight loss, fatigue, fever or other systemic complaints and no family history of breast cancer. Significant past medical history included treatment for an extramedullary retrosternal plasmacytoma 5-years prior this admission. At the time of the initial work-up for the retrosternal mass, immunoelectrophoresis showed no evidence for hyperproteinemia or paraproteinemia. Whole body bone scan was negative and a bone marrow biopsy revealed less than 5% of plasma cells. Therefore, multiple myeloma was excluded by nuclear medicine, laboratory and histology studies. The patient underwent radiation therapy (40 Gy with fraction size of 200 cGy delivered over 4 weeks) followed by chemotherapy with cyclophosphamide, cisplatin and prednisolone. The patient was followed by laboratory tests, chest roentgenography, and computed tomography annually. A bone scintigraphy was carried out after 2 years and showed no uptake patient was thereafter lost to follow-up. Five years after initial diagnosis of extramedullary plasmacytoma the patient presented with bilateral breast masses. Physical examination revealed a 3.5 cm × 2.5 cm, mass in the upper inner quadrant of the right breast and a similar 5.0 cm × 4.5 cm mass in the lower inner quadrant of the left breast. No asymmetry, skin dimpling or signs of inflammation were present. There was no axillary or supraclavicular lymphadenopathy. Mammography confirmed a well-defined 3.2 cm oval-shaped mass in the upper inner quadrant of the right breast, and a lobulated 5.5 cm density in lower inner quadrant of the left breast without any tissue distortion, inflammation and fibrotic reaction.(Figure 1) There were no microcalcification and satellite lesions. These masses were solid and hypoechoeic with multiple septations in sonography. Figure 1 Mammography of the patients' breasts (A: mediolateral oblique, view B: craniocaudal view) Excisional biopsy of the masses revealed a 5.0 (left) and 3.0 (right) well-defined, capsulated gritty mass surrounded by normal breast tissue. There was no extension from the capsulated masses to pectoral muscles or chest wall. Histopathological examination showed high-grade tumors composed of immature and mature plasma cells. Mitosis, necrosis, nuclear pleomorphism and binucleated and multinucleated plasma cells were seen. (Figure 2) Additional studies such as serum protein electrophoresis and immunoelectrophoresis were normal. No Bence Jones or other M components were detected in the urine. Skeletal surveys (Tc99 bone scan and skull and pelvic X-rays) did not show any pathological changes. There was no evidence of anemia, hypercalcemia or renal insufficiency. However, the patient refused a second bone marrow biopsy. Figure 2 Photomicrograph showing nuclear pleomorphism, binucleated and multinucleated plasma cells with enlarged nucleoli (Hematoxylin & Eosin). Immunohistochemical studies were performed on the paraffin embedded tissues to determine if the infiltrate had monoclonal character. The tumor cells were diffusely and strongly positive for lambda chains but negative for kappa chains. (Figure 3) Figure 3 Lambda and kappa immunohistochemical stain showing strong and diffuse positivity for lambda (a) and negativity for kappa (b). The tumor cells were weakly positive for monoclonal mouse anti human placental V538C, and plasma cell markers (CD138). Nuclear prognostic marker (Ki67) showed 50% to 80% nuclear expression indicative of high proliferative activity and suggesting a plasmacytic tumor with anaplastic components (Figure 4). Other immunohistochemical stains including CD21, cytokeratin, S100, and HMB45 were negative. Figure 4 Ki67 immunohistochemical stain showing 50–80% nuclear positivity A retrospective microscopic review of the mediastinal mass showed similar morphology to the breast tumor. Hence, the histological diagnosis of recurrent plasmacytoma was made. The patient was treated with oral Melfalan and Prednisone. The patient has been disease free for twenty months after treatment and has showed no evidence of recurrence in the mediastinum, breast or any other region. Discussion Primary soft tissue extramedullary plasmacytoma (SEP) is uncommon and is defined as a malignant tumor of plasma cells arising in the soft tissue in the absence of bone involvement. It can occur in any organ as a solitary form of plasma cell neoplasm [2]. Although SEP can arise throughout the body, almost 90% of the cases arise in the head and neck areas, most commonly in the upper respiratory tract including the nasal cavity, para nasal sinuses, oropharynx, salivary glands and larynx [3-7]. Several other sites can rarely be involved, including testis, bladder, urethra, breast, ovary, lung, pleura, thyroid, orbit, brain and skin tissues (1, 8–17). Approximately forty-five cases of breast plasmacytoma have been reported in published literature since 1928 (1, 2, 18–21). More than half of the lesions were unilateral (66%), with the majority of the cases occurring in the setting of multiple myeloma (77%) (1, 2, 18–20, 22). When plasmacytoma originates from soft tissues, like the case presented here, the disease is usually associated with a relatively mild clinical behavior and long survival, suggesting that it is a truly different disease entity compared to other plasma cell tumors [23]. Dimopoulos et al (1999) reported that solitary extramedullary (soft tissue) plasmacytomas (SEP) are less common than solitary bone plasmocytoma (SBP), and have a better prognosis as the majority can be cured by local radiotherapy [24]. Liebross et al (1999) reported local recurrence rates of less than 5% after radiotherapy [6]. Mayr et al (1990) noted that the risk of distant relapse is more than 30%, which is significantly less than that seen with SBP [25]. Progressive disease may accrue as multiple myeloma, SBP or involvement of lymph nodes, skin or subcutaneous tissues. Its recurrence, if any, tends to be within 2–3 years of initial diagnosis. The reported ten year survival rate is at least 66% [3,7]. Involvement of the breast with SEP is uncommon and may occur either as a solitary primary tumor or as evidence of disseminated multiple myeloma [19]. Our patient was referred with bilateral breast masses. In an approach to a patient with bilateral breast masses, differential diagnosis includes: fibroadenomas, complex cysts, metastasis, lymphoma, synchronous breast cancer, focal fibrosis, fat necrosis, abscess, and phyllodes tumor [20]. Metastatic lesions of the breast from extramammary neoplasms are rare and in larger studies have been reported to constitute 0.4 to 2% of all breast malignancies. The most common are lymphomas and other tumors of hematological origin [26]. The striking feature in this case was the recurrence of an isolated plasmacytoma (which was treated successfully five year prior) in both breasts. As approximately 20% of patients who present with isolated extramedullary plasmacytoma will eventually develop multiple myeloma, close follow-up is strongly recommended [1,22]. Our patient did not have any prior breast aspiration cytology. There are many tumors, which may present with plasmacytoid appearance on aspiration cytology. Tumors such as small ductal carcinoma and lobular carcinoma of breast, metastatic carcinoid, metastatic melanoma and some lymphomas may represent with uncohesive group of cells with eccentric nuclei resembling plasma cells. Hence, a plasmacytoma (with anaplastic plasma cells) may be readily mistaken for carcinoma (or other undifferentiated neoplasm) not only clinically, but also on cytological examination. This would justify excision biopsy and the use of an extended immunohistochemical panel to include such markers as cytokeratin and S-100 in the assessments. To help the cytopathologist avoid misinterpretation, clinical history and presentation are extremely helpful. Most of the errors in histopathology and cytopathology diagnosis occur when pathologist is not aware of medical history of the patient and unusual clinical presentation. This case emphasizes the importance of distinguishing a plasmacytoma of the breast from primary mammary carcinomas and other benign lesions to avoid unnecessary surgery and provide the appropriate treatment and adjuvant therapy. Authors' Contributions AK carried out excision biopsies and drafted the manuscript. MJZ did the histopathological examination and contributed to the pathological content of the manuscript. SKR is the pathologist who confirmed the diagnosis and prepared the immunohistochemical stains and illustrations. He also contributed to pathological content of the manuscript MN followed-up the patient and contributed to the manuscript preparation All authors read and approved the final manuscript. Acknowledgments Patient's permission was obtained for publishing her case records. Authors also wish to thanks Dr Reza Navipour, Center for Research Development, Imam Hospital, Tehran University of Medical Sciences for his help in accessing the patient's medical documents. ==== Refs De Chiara A Losito S Terracciano L Di Giacomo R Iaccarino G Rubolotta MR Primary plasmacytoma of the breast Arch Pathol Lab Med 2001 125 1078 1080 11473462 Van Nieuwkoop C Giard RW Veen HF Dees A Extramedullary plasmacytoma of the breast simulating breast cancer Neth J Med 2001 58 174 176 11325494 10.1016/S0300-2977(01)00091-2 Brinch L Hannisdal E Abrahamsen AF Kvaloy S Langholm R Extramedullary plasmacytomas and solitary plasma cell tumors of bone Eur J Haematol 1990 44 132 135 2318296 Wax MK Yun KJ Omar RA Extramedullary plasmacytomas of the head and neck Otolaryngol Head Neck Surg 1993 109 877 885 8247569 Susnerwala SS Shanks JH Banerjee SS Scarffe JH Farrington WT Slevin NJ Extramedullary plasmacytoma of the head and neck region: clinicopathological correlation in 25 cases Br J Cancer 1997 75 921 927 9062417 Liebross RH Ha CS Cox JD Weber D Delasalle K Alexanian R Clinical course of solitary extramedullary plasmacytoma Radiother Oncol 1999 52 245 249 10580871 10.1016/S0167-8140(99)00114-0 Galieni P Cavo M Pulsoni A Avvisati G Bigazzi C Neri S Caliceti U Benni M Ronconi S Lauria F Clinical outcome of extramedullary plasmacytoma Haematologica 2000 85 47 51 10629591 Cavanna L Fornari F Civardi G Di Stasi M Sbolli G Foroni R Voltolini F Buscarini L Extramedullary plasmacytoma of the testicle. Sonographic appearance and ultrasonically guided biopsy Blut 1990 60 328 330 2198076 Rubin J Johnson JT Killeen R Barnes L Extramedullary plasmacytoma of the thyroid associated with a serum monoclonal gammopathy Arch Otolaryngol Head Neck Surg 1990 116 855 859 2363927 Matsumiya K Kanayama Y Yamaguchi S Ueyama Y Iwasaki M Osafune M Extramedullary plasmacytoma (EMP) of urinary bladder Urology 1992 40 67 70 1621316 10.1016/0090-4295(92)90440-8 Nonomura A Mizukami Y Shimizu J Oda M Watanabe Y Kamimura R Takashima T Kitagawa M Primary extramedullary plasmacytoma of the lung Intern Med 1992 31 1396 1400 1300176 Wong KF Chan JK Li LP Yau TK Lee AW Primary cutaneous plasmacytoma – report of two cases and review of the literature Am J Dermatopathol 1994 16 392 397 7978068 Adkins JW Shields JA Shields CL Eagle RC JrFlanagan JC Campanella PC Plasmacytoma of the eye and orbit Int Ophthalmol 1996 20 339 343 9237136 Fischer C Terpe HJ Weidner W Schulz A Primary plasmacytoma of the testis. Case report and review of the literature Urol Int 1996 56 263 265 8776829 Tuting T Bork K Primary plasmacytoma of the skin J Am Acad Dermatol 1996 34 386 390 8655732 Emery JD Kennedy AW Tubbs RR Castellani WJ Hussein MA Plasmacytoma of the ovary: a case report and literature review Gynecol Oncol 1999 73 151 154 10094897 10.1006/gyno.1998.5246 Muscardin LM Pulsoni A Cerroni L Primary cutaneous plasmacytoma: report of a case with review of the literature J Am Acad Dermatol 2000 43 962 965 11044834 10.1067/mjd.2000.103997 Cangiarella J Waisman J Cohen JM Chhieng D Symmans WF Goldenberg A Plasmacytoma of the breast: A report of two cases diagnosed by aspiration biopsy Acta Cytol 2000 44 91 94 10667168 Lamy O Von Bremen K Burckhardt P Breast plasmacytoma Leuk Lymphoma 2000 37 611 615 11042522 Brem RF Revelon G Willey SC Gatewood OM Zeiger MA Bilateral plasmacytoma of the breast: a case report Breast J 2002 8 393 395 12390367 10.1046/j.1524-4741.2002.08613.x Harmouche H Cahen-Doidy L Bourgarit A Farge D Sereni D Breast plasmacytoma. A new case Rev Med Interne 2002 23 207 209 11876068 10.1016/S0248-8663(01)00541-0 Dagli M Demirer T Ayli M Haznedar R Gunel N Sucak G Fen T Moran M Ustun T Aybirdi C Cavusoglu M Dincer S Uysal A plasmacytoma in bilateral breast Turk J Haematol 2002 19 481 483 Wiltshaw E The natural history of extramedullary plasmacytoma and its relation to solitary myeloma of bone and myelomatosis Medicine 1976 55 217 238 1272069 Dimopoulos MA Kiamouris C Moulopoulos LA Solitary plasmacytoma of bone and extramedullary plasmacytoma Hematol Oncol Clin North Am 1999 13 1249 1257 10626148 Mayr NA Wen BC Hussey DH Burns CP Staples JJ Doornbos JF Vigliotti AP The role of radiation therapy in the treatment of solitary plasmacytomas Radiother Oncol 1990 17 293 303 2343147 Domanski HA Metastases to the breast from extra mammary neoplasms. A report of six cases with diagnosis by fine needle aspiration cytology Acta cytologica 1996 40 1293 1300 8960043

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World J Surg Oncol. 2004 Aug 31; 2:29

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World J Surg Oncol

10.1186/1477-7819-2-29

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-611532769310.1186/1477-7827-2-61ResearchAntimicrobial actions of the human epididymis 2 (HE2) protein isoforms, HE2alpha, HE2beta1 and HE2beta2 Yenugu Suresh [email protected] Katherine G [email protected] Frank S [email protected] Susan H [email protected] Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7500, USA2004 24 8 2004 2 61 61 16 6 2004 24 8 2004 Copyright © 2004 Yenugu et al; licensee BioMed Central Ltd.2004Yenugu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The HE2 gene encodes a group of isoforms with similarities to the antimicrobial beta-defensins. We demonstrated earlier that the antimicrobial activity of HE2 proteins and peptides is salt resistant and structure dependent and involves permeabilization of bacterial membranes. In this study, we further characterize the antimicrobial properties of HE2 peptides in terms of the structural changes induced in E. coli and the inhibition of macromolecular synthesis. Methods E. coli treated with 50 micro g/ml of HE2alpha, HE2beta1 or HE2beta2 peptides for 30 and 60 min were visualized using transmission and scanning electron microscopy to investigate the impact of these peptides on bacterial internal and external structure. The effects of HE2alpha, HE2beta1 and HE2beta2 on E. coli macromolecular synthesis was assayed by incubating the bacteria with 2, 10 and 25 micro g/ml of the individual peptides for 0–60 min and measuring the incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein. Statistical analyses using Student's t-test were performed using Sigma Plot software. Values shown are Mean ± S.D. Results E. coli treated with HE2alpha, HE2beta1 and HE2beta2 peptides as visualized by transmission electron microscopy showed extensive damage characterized by membrane blebbing, thickening of the membrane, highly granulated cytoplasm and appearance of vacuoles in contrast to the smooth and continuous membrane structure of the untreated bacteria. Similarly, bacteria observed by scanning electron microscopy after treating with HE2alpha, HE2beta1 or HE2beta2 peptides exhibited membrane blebbing and wrinkling, leakage of cellular contents, especially at the dividing septa, and external accumulation of fibrous materials. In addition, HE2alpha, HE2beta1 and HE2beta2 peptides inhibited E. coli DNA, RNA and protein synthesis. Conclusions The morphological changes observed in E. coli treated with epididymal HE2 peptides provide further evidence for their membrane dependent mechanism of antibacterial action. HE2 C-terminal peptides can inhibit E. coli macromolecular synthesis, suggesting an additional mechanism of bacterial killing supplementary to membrane permeabilization. ==== Body Introduction Antimicrobial proteins and peptides are widely expressed in both plants and animals. A variety of natural antibiotics belonging to different classes such as defensins, cathelicidins, cercopins and protease inhibitors [1] are found in epithelial tissues of organs that are most likely exposed to pathogens. Among them, the most studied in humans are the defensins, which are broadly classified into three types viz alpha, beta and theta defensins depending on their disulfide bonding, tissue distribution and genomic organization. They exhibit broad spectrum antimicrobial activity [2-5], thus may form an important component of the innate immune system. Antimicrobial proteins and peptides including defensins are generally cationic in nature [6] and are believed to exert their bactericidal effect by permeabilizing the bacterial membranes by forming pores [7], thinning the membrane [8], or by destabilizing the membrane bilayer [9]. In addition to membrane permeabilization, antimicrobial proteins and peptides kill bacteria by inhibition of macromolecular biosynthesis [10-12] and/or interacting with specific vital components inside the bacteria [13,14]. In the epididymis, a major organ of the male reproductive tract, immature sperm released from the testis undergo sequential maturation to acquire forward motility and fertilizing ability. A wide variety of proteins including antimicrobial proteins released into the lumen of epididymis bind sperm and are thought to play an important role in epididymal immunity in addition to their role in sperm maturation [15]. Examples of antimicrobial proteins reported in the male reproductive tract include human cationic antimicrobial protein (hCAP18, a cathelicidin) [16], defensins [17-20], the epididymal β-defensin member Bin1b [21], cystatins [22,23], lactoferrin [24] seminalplasmin [25] and seminogelin-derived peptides [26]. Earlier we identified and characterized the sperm binding epididymal proteins of the HE2 family [27], which show homology to the antimicrobial β-defensins. The HE2 gene located on chromosome 8p23 within the β-defensin gene cluster, encodes a series of isoforms containing identical proregions joined to different C-terminal peptides [27]. Among them, HE2β1 conserves the characteristic β-defensin-like six-cysteine motif (Figure 1). Furthermore, like the β-defensins, HE2 C-terminal peptides are cleaved from their proregions by a furin-like proprotein convertase and these peptides are reported to exist in the epididymal epithelium, luminal fluid and the seminal plasma [28]. We demonstrated the antimicrobial activity of HE2α, HE2β1 and HE2β2 proteins and their C-terminal peptides [29] and the epididymis specific defensin DEFB118 [30] against E. coli. Their antimicrobial activities are structure dependent and salt tolerant and their mechanism of action involves interacting with and permeabilizing bacterial membranes. However, structural evidence for the membrane changes in E. coli induced by these peptides is still lacking. Further, it is not still clear whether bacterial killing by HE2 peptides involves only membrane permeabilization or whether the peptides interact with specific targets inside the bacteria to inhibit metabolic processes as reported for other antimicrobial proteins is not yet demonstrated. In this study, using transmission and scanning electron microscopy, we provide further evidence that HE2 peptides induce significant structural changes in E. coli consistent with their membrane dependent mechanism of action as reported earlier. Further, we show that HE2 peptides inhibit E. coli DNA, RNA and protein synthesis suggesting that their antimicrobial action may also involve targets inside the bacteria as well as membrane permeabilization. Figure 1 Amino acid sequence alignment of epididymal HE2 peptides with human β-defensins 1 and 2. Amino acid sequence shown in blue corresponds to the C-terminal peptides used in this study. The characteristic β-defensin six cysteine motif is represented in red. represents the cleavage site where the full length proteins are cleaved to release the C-terminal peptides. Methods Recombinant peptide preparation and synthesis HE2α and HE2β2 C-terminal peptides were synthesized at the Peptide Synthesis Facility, University of North Carolina, Chapel Hill by standard f-moc solid phase procedures using Rainin symphony multiple peptide synthesizer (Rainin Instrument, Woburn, MA). The purified peptides eluted as single peaks upon reverse phase high performance liquid chromatography (HPLC) and were further demonstrated to have their corresponding molecular weight by MALDI-TOF mass spectrometry. HE2β1 C-terminal peptide was expressed in E. coli and purified as described previously [29]. Briefly, E. coli strain M15 (pREP4) was transformed with pQE30 vector (Qiagen, Valencia, CA, U.S.A) containing cDNA that codes for HE2β1 C-terminal peptide. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside for 1 h at 37°C and the His-tagged recombinant peptide was purified using nickel-nitrilotriacetate agarose column (Qiagen, Valencia, CA, U.S.A). To avoid baseline expression of the protein prior to induction, 1% glucose was maintained in the bacterial medium and the induction time was kept to a minimum (1 h) to minimize the toxic effects of the peptide on E. coli. The peptide was dialyzed extensively against 10 mM sodium phosphate (pH 7.4) to remove urea. Transmission electron microscopy E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with 50 μg/ml HE2α, HE2β1 or HE2β2 for 30 and 60 min. After incubation, bacterial cells were washed with 10 mM sodium phosphate buffer (pH 7.4) and fixed with an equal volume of 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, followed by centrifugation at 1000 rpm for 10 minutes to concentrate the cells in a pellet. The fixed samples were stored overnight to several days at 4°C in the fixative solution. The pellet was rinsed in 0.1 M sodium cacodylate buffer several times, and post-fixed with a combination of 1.25% potassium ferrocyanide and 1% buffered osmium tetroxide for one hour at room temperature. Following dehydration with a graded series of ethanols (30–100%) and two changes of propylene oxide, the cell pellet was infiltrated and embedded in PolyBed 812 epoxy resin (Polysciences, Inc., Warrington, PA). Ultra thin sections (70 nm) were cut and mounted on copper grids followed by post staining with 4% uranyl acetate and 0.4% lead citrate. The sections were examined and photographed at an accelerating voltage of 80 kV using a LEO EM 910 transmission electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) equipped with a Gatan BioScan digital camera (Gatan, Inc., Pleasanton, CA). Scanning electron microscopy The structural changes induced by HE2 peptides on E. coli were studied using scanning electron microscopy as described earlier [30]. Bacterial cells suspended in 10 mM sodium phosphate buffer (pH 7.4) after treating with 50 μg/ml of HE2 peptide were fixed with an equal volume of 4% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4. Immediately following the addition of the fixative solution, the sample tube was mixed by gently inverting the tube up and down for several minutes to prevent clumping of the cells. The fixed samples were stored overnight to several days at 4°C in the fixative solution. Using a microanalysis vacuum filter holder (Fisher Scientific, Suwanee, GA) and a 0.1 μm polycarbonate membrane filter (Poretics Corporation, Livermore, CA), the suspended fixed cells were vacuum-filtered onto the membrane substrate, rinsed with 0.15 M sodium phosphate buffer, and dehydrated through a graded series of ethanols (30–100%). During the entire filtration, rinsing, and dehydration process, the cells were kept covered with fluid to prevent air drying. The filters were transferred in 100% ethanol to a critical point dryer (Balzers CPD-020, Bal-Tec AG, Vaduz, Liechtenstein), and dried using carbon dioxide as the transition solvent. The filters were mounted on aluminum specimen supports with carbon adhesive tabs, and coated with a 15 nm thickness of gold-palladium metal (60:40 alloy) using a Hummer X sputter coater (Anatech, Ltd., Alexandria, VA). Samples were examined with a Cambridge Stereoscan 200 scanning electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) using an accelerating voltage of 20 kV. Macromolecular synthesis The effects of HE2 peptides on E. coli DNA, RNA and protein synthesis were studied as functions of incorporation of the radioactive precursors [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine respectively as described [30]. 1 × 106 mid-log phase E. coli resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with varying concentrations of HE2 peptides and 2.5 μl/ml of either [methyl-3H]thymidine (20 Ci/mmol), [5-3H]uridine (25.5 Ci/mmol) or L-[4,5-3H(N)]leucine (59.5 Ci/mmol) for different time periods. After incubation, bacterial suspensions were added to 10% ice-cold trichloroacetic acid and allowed to stand in ice for 40 min. Samples were then collected on 2.4 cm GF/C glass microfiber filters (Fisher Scientific, Pittsburgh, PA) using vacuum filtration and washed thoroughly with 5% TCA and 70% ethanol. The filters were then dried and placed in scintillation vials containing 5 ml of EcoScint scintillation cocktail (National Diagnostics, Atlanta, GA) and counts were obtained in a LKB 1214 Rackbeta liquid scintillation counter (LKB WALLACE, Turku, Finland) for 1 min for each filter. Statistical analyses using Student's t-test were performed using Sigma Plot software (SPSS Inc., Chicago, IL). Values shown are Mean ± S.D. Results Transmission electron microscopy Transmission electron microscopy revealed striking structural alterations in E. coli exposed to HE2 peptides. The three images shown for each treatment were documented in different fields of view and are intended to represent the range of responses seen in the bacteria. In contrast to the smooth continuous double membrane structure clearly visible in untreated bacteria (Fig. 2A,2B,2C), the outer membranes of bacteria treated with 50 μg/ml HE2α peptide for 30–60 min showed thickening and the protrusion of irregular blebs. The inner membrane was indistinct in many regions after 30 min and the cytoplasm was retracting from the outer membrane (Fig. 3A,3B,3C). After 60 min of HE2α treatment, the inner membrane was difficult to discern and fibrous and granular material, presumably cell contents appeared to exude from the damaged membranes (Fig. 3D,3E,3F). Treatment with 50 μg/ml HE2β1 peptide for 30 min resulted in numerous mushroom shaped blebs and retraction of cytoplasm (Fig. 4A,4B,4C) and by 60 min, these bacteria appeared to lose cell contents particularly at the division septa (Fig. 4D,4E,4F). Similarly, the HE2β2-treated bacteria showed loss of the double membrane structure, formation of blebs and outer membrane roughening (Fig. 5A,5B,5C). By 60 min, numerous large vacuoles accumulated, the cytoplasm was extensively granulated and retracted from the outer membrane and cell contents appeared to escape at division septa (Fig. 5D,5E,5F). The peptides appeared to induce structural changes specific to each peptide besides the morphological changes that were generally observed. HE2α peptide caused membrane thickenings, which was not observed with the other two peptides. Similarly, HE2β1 peptide caused retraction of cytoplasm when treated for 30 min, whereas HE2β2 peptide induced retraction of cytoplasm after 60 min incubation. Formation of vacuoles was more evident upon treating E. coli with HE2β2 peptide. Figure 2 Transmission electron micrographs of untreated E. coli showing a smooth continuous double membrane structure. Figure 3 E. coli treated with 50 μg/ml HE2α peptide for 30 min (A-C) and 60 min (D-F) visualized by transmission electron microscopy showed membrane thickening and blebbing with subsequent leakage of cellular contents. Figure 4 Transmission electron micrographs showing cytoplasmic retraction and extensive granulation of E. coli treated with 50 μg/ml HE2β1 peptide for 30 min (A-C) and 60 min (D-F). Figure 5 Incubation of E. coli with 50 μg/ml HE2β2 peptide for 30 min (A-C) and 60 min (D-F) show discontinuous membrane structure with extensive vacuole formation. Cellular contents appear to leak at the dividing septa. Scanning electron microscopy E. coli treated with HE2 peptides were observed using scanning electron microscopy to gain further insights into the membrane effects. The three images shown for each treatment were documented in different fields of view and are intended to represent the range of responses seen in the bacteria. Untreated bacterial cells had normal and smooth surface morphology (Fig. 6A,6B,6C). Bacteria treated with HE2α (Fig. 7A,7B,7C,7D,7E,7F), HE2β1 (Fig. 8A,8B,8C,8D,8E,8F) or HE2β2 (Fig. 9A,9B,9C,9D,9E,9F) peptides showed pronounced changes in their morphology consistent with the changes observed using transmission electron microscopy. E. coli treated with the HE2 peptides for 30–60 min showed pronounced wrinkling, surface roughening and blebbing of the membrane. A majority of the cells appeared to have lost their bacterial membrane integrity. The fibrous material and cellular debris, possibly arising due to leakage and cell lysis accumulated particularly at the dividing septa. Figure 6 Scanning electron micrographs of untreated E. coli revealing a smooth membrane surface morphology. Figure 7 E. coli treated with 50 μg/ml HE2α peptide for 30 min (A-C) and 60 min (D-F) visualized by scanning electron microscopy show membrane blebbing and leakage of cellular contents. Figure 8 Membrane wrinkling and blebbing were evident in E. coli treated with 50 μg/ml HE2β1 peptide for 30 min (A-C) and 60 min (D-F). Figure 9 Scanning electron micrographs of E. coli treated with 50 μg/ml HE2β2 peptide for 30 min (A-C) and 60 min (D-F). Loss of bacterial membrane integrity due to surface blebbing and wrinkling was evident. Macromolecular synthesis To investigate whether HE2 peptides affect macromolecular synthesis of E. coli, the incorporation of radioactive precursors viz [methyl-3H]thymidine, [5-3H]uridine and L-[4,5-3H(N)]leucine into DNA, RNA and protein was studied in the presence of 2–25 μg/ml peptides. A dose and time dependent inhibition of DNA synthesis by HE2α peptide was observed (Fig. 10A). 2 μg/ml HE2α peptide inhibited DNA synthesis after 60 min incubation, whereas 10 and 25 μg/ml significantly inhibited DNA synthesis after 20 min incubation (Fig. 10A). RNA synthesis was not inhibited by 2 μg/ml HE2α peptide, whereas inhibition was observed with 10 and 25 μg/ml concentrations (Fig. 10B). No significant inhibition of protein synthesis by HE2α peptide was observed at any of the concentrations tested (Fig. 10C). Figure 10 Effect of HE2α peptide on macromolecular synthesis in E. coli. A, [methyl-3H]thymidine incorporation into DNA. B, [5-3H]uridine incorporation into RNA. C, L-[4,5-3H(N)]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. *, P < 0.05-0.01, **, P < 0.01-0.001, ***, P < 0.001 compared to 0 μg/ml at the corresponding time point. In the case of HE2β1 peptide, 2 μg/ml dose did not inhibit DNA synthesis, whereas 10 and 25 μg/ml concentrations showed significant inhibition (Fig. 11A) after a 20 min incubation. Similarly, significant inhibition of RNA synthesis was not observed with 2 μg/ml. However, 10 and 25 μg/ml concentrations inhibited RNA after 20 min (Fig. 11B) and protein synthesis (Fig. 11C) after a 60 min incubation. Figure 11 Effect of HE2β1 peptide on macromolecular synthesis in E. coli. A, [methyl-3H]thymidine incorporation into DNA. B, [5-3H]uridine incorporation into RNA. C, L-[4,5-3H(N)]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. *, P < 0.05-0.01, **, P < 0.01-0.001, ***, P < 0.001 compared to 0 μg/ml at the corresponding time point. Inhibition of E. coli DNA synthesis by HE2β2 peptide was dose and time dependent. Significant inhibition of DNA synthesis was observed after a 60 min incubation with 2 μg/ml HE2β2 peptide, whereas the inhibition was observed at a earlier time point with 10 and 25 μg/ml concentrations (Fig. 12A). However, RNA synthesis was not inhibited by 2 μg/ml HE2β2 peptide, whereas 10 and 25 μg/ml doses were effective after 40 and 10 min incubations respectively (Fig. 12B). Protein synthesis was inhibited only with 10 and 25 μg/ml HE2β2 peptide after a 60 min incubation (Fig. 12C). It appears that HE2 peptides inhibit DNA synthesis to a greater extent than RNA and protein synthesis, suggesting that DNA synthesis may be the sensitive target for antimicrobial action after membrane permeabilization. Figure 12 Effect of HE2β2 peptide on macromolecular synthesis in E. coli. A, [methyl-3H]thymidine incorporation into DNA. B, [5-3H]uridine incorporation into RNA. C, L-[4,5-3H(N)]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. *, P < 0.05-0.01, **, P < 0.01-0.001, ***, P < 0.001 compared to 0 μg/ml at the corresponding time point. Discussion Earlier we demonstrated that HE2 proteins and their C-terminal peptides exhibit salt tolerant and structural dependent antimicrobial activities and their mechanism involved permeabilization of both outer and inner bacterial membranes [29]. In this study, structural changes induced in E. coli by epididymal HE2α, HE2β1 and HE2β2 peptides as visualized by transmission and scanning electron microscopy provide further evidence of the membrane dependent mechanism of bacterial killing. Such structural changes induced in E. coli by other antimicrobial proteins and peptides were reported previously. Membrane thickening as shown in Fig 3A,3B,3C,3D,3E,3F was reported in E. coli treated with human neutrophil peptides 1 and 2 (defensins) [14]. Similarly, retraction of cytoplasm and the appearance of vacuoles as shown in Fig 4A,4B,4C,4D,4E,4F were reported for E. coli treated with synthetic peptides of the antimicrobial protein apolipoprotein A-II [31]. Highly granular cytoplasm with discontinuous membrane was reported for E. coli treated with the antimicrobial peptide tigerinin-1 [32] similar to the changes shown in Fig. 5A,5B,5C,5D,5E,5F. Scanning electron micrographs of E. coli treated with HE2 peptides also revealed striking structural changes in their morphology. HE2 peptides caused membrane wrinkling, blebbing and leakage of fibrous material primarily at the dividing septa in E. coli. Such structural changes shown in Fig. 7,8,9 were earlier reported for other antimicrobial proteins viz the cathelicidin-derived peptide SMAP-29 [33], temporin-L [34], salmon antimicrobial protein [35] and the epididymal proteins ESC42 (DEFB118) [30] and EPPIN [36]. An interesting observation in this study is the leakage of fibrous material primarily at the dividing septa. It is known that cell division in E. coli involves annular constriction of all layers of the cell envelope and synthesis and assembly of new septal materials [37]. It is possible that during this dynamic remodeling process, the region of division septum formation to be particularly vulnerable to attack by antibacterial proteins. The mechanism of action of antimicrobial proteins is primarily thought to be membrane dependent involving membrane permeabilization and disruption. Structural characteristics of antimicrobial peptides tend to play an important role in their mechanism of action. For example, β-defensins are cationic in nature and with β-sheet rich amphipathic structures stabilized by the three disulfide motif [38]. The cationic nature of β-defensins favors them to bind to and disrupt target membranes that are rich in anionic phospholipids. Similarly, HE2α, HE2β1 and HE2β2 peptides are cationic in nature with basic pIs. Our three dimensional structural analysis of HE2β1 peptide revealed that it is rich in β-sheet structure and its tertiary structure presents regional concentrations of basic and hydrophobic amino acids similar to β-defensins [29]. Such structural characteristics of HE2 peptides which resemble to those of β-defensins suggest that they bind to and disrupt the anionic target membranes and mediate bacterial killing similar to β-defensins. However, alternate mechanisms of antimicrobial action such as inhibition of macromolecular synthesis [10-12] and interaction with specific targets inside the bacterial cells [13,14] are proposed. HE2 peptides at 10 and 25 μg/ml concentrations inhibited DNA, RNA and protein synthesis suggesting that their antimicrobial action may include interference with metabolic functions of E. coli. Inhibition of macromolecular synthesis was reported for bactenectins [39], human neutrophil peptide-1 [40], pleurocidin derived peptides [41] and the epididymal defensin DEFB118 [30]. In this study, it appears that HE2 peptides were more effective in inhibiting the incorporation of [methyl-3H]thymidine than [5-3H]uridine and L-[4,5-3H(N)]leucine, suggesting DNA synthesis is more sensitive to their antimicrobial action. It is possible that in bacteria that are extensively damaged by HE2 peptides, inhibition of macromolecular synthesis may result simply from the total breakdown of the cells. However the electron micrographs show that only some bacteria appear to be exuding cell contents after the 30 minute treatment. Thus during the first 10–20 minutes exposure to HE2 peptides, some peptides may be entering through pores too small for major cytoplasmic release. The early inhibition of DNA and RNA synthesis in bacteria where little loss of cell contents has occurred, may result from specific interaction of the synthetic machinery with HE2 peptides. Further studies are required to identify specific molecular targets within the bacteria and to establish whether HE2 interactions with these targets can be beneficial to the host by slowing bacterial proliferation. Increasing recognition of the ability of a number of proteins on the sperm surface to kill bacteria has led to the proposal that they may defend against microbial attack in both the male and female reproductive tracts. The cathelicidin hCAP18 on sperm is processed by the prostate-derived protease, gastricsin to release the active peptide ALL-38 and is found in the female reproductive tract after intercourse [42]. A member of the β-defensin family, DEFB126 also appears to have a role in fertility as a capacitation factor on sperm [43]. Similarly, the rat epididymis specific β-defensin Bin1b, appears to play an important role in sperm maturation [44]. Thus, these defense proteins may enhance the probability of successful fertilization in addition to helping prevent the spread of sexually transmitted diseases. Conclusions In conclusion, we report that the epididymal antimicrobial peptides HE2α, HE2β1 and HE2β2 induce striking morphological changes in E. coli consistent with their membrane dependent mechanism of action [29]. In addition to membrane permeabilization, their antimicrobial mechanism involves inhibition of E. coli DNA, RNA and protein synthesis. Author's contributions SY performed the electron microscopy studies, radioactive incorporation assays and wrote majority of the manuscript. KGH prepared the recombinant peptides. SHH and FSF supervised and coordinated the work and the preparation of the manuscript. All authors read, commented upon and approved the final manuscript. Acknowledgements We thank Victoria Madden, Microscopy Services Laboratory (MSL), University of North Carolina for her assistance with the electron microscopy. This work was supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of Contraceptive Research and Development or Consortium for Industrial Collaboration in Contraceptive Research. This work is also supported by NIH Grants R37-HD04466, by National Institute of Child Health and Human development/NIH through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by the Fogarty International Center Training and Research in Population and Health Grant D43TW / HD00627. ==== Refs Ganz T Antimicrobial polypeptides J Leukoc Biol 2004 75 34 38 12960278 10.1189/jlb.0403150 Bastian A Schafer H Human alpha-defensin 1 (HNP-1) inhibits adenoviral infection in vitro Regul Pept 2001 101 157 161 11495691 10.1016/S0167-0115(01)00282-8 Daher KA Selsted ME Lehrer RI Direct inactivation of viruses by human granulocyte defensins J Virol 1986 60 1068 1074 3023659 Schibli DJ Hunter HN Aseyev V Starner TD Wiencek JM McCray P. B., Jr. Tack BF Vogel HJ The solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus J Biol Chem 2002 277 8279 8289 11741980 10.1074/jbc.M108830200 Lehrer RI Ganz T Defensins of vertebrate animals Curr Opin Immunol 2002 14 96 102 11790538 10.1016/S0952-7915(01)00303-X Zasloff M Antimicrobial peptides of multicellular organisms Nature 2002 415 389 395 11807545 10.1038/415389a Matsuzaki K Magainins as paradigm for the mode of action of pore forming polypeptides Biochim Biophys Acta 1998 1376 391 400 9804997 10.1016/S0304-4157(98)00014-8 Heller WT Waring AJ Lehrer RI Harroun TA Weiss TM Yang L Huang HW Membrane thinning effect of the beta-sheet antimicrobial protegrin Biochemistry 2000 39 139 145 10625488 10.1021/bi991892m Gazit E Boman A Boman HG Shai Y Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles Biochemistry 1995 34 11479 11488 7547876 Park CB Kim HS Kim SC Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions Biochem Biophys Res Commun 1998 244 253 257 9514864 10.1006/bbrc.1998.8159 Subbalakshmi C Sitaram N Mechanism of antimicrobial action of indolicidin FEMS Microbiol Lett 1998 160 91 96 9495018 10.1016/S0378-1097(98)00008-1 Boman HG Agerberth B Boman A Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine Infect Immun 1993 61 2978 2984 8514403 Otvos L., Jr. O I Rogers ME Consolvo PJ Condie BA Lovas S Bulet P Blaszczyk-Thurin M Interaction between heat shock proteins and antimicrobial peptides Biochemistry 2000 39 14150 14159 11087363 10.1021/bi0012843 Lehrer RI Barton A Daher KA Harwig SS Ganz T Selsted ME Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity J Clin Invest 1989 84 553 561 2668334 Hall SH Hamil KG French FS Host defense proteins of the male reproductive tract J Androl 2002 23 585 597 12185087 Malm J Sorensen O Persson T Frohm-Nilsson M Johansson B Bjartell A Lilja H Stahle-Backdahl M Borregaard N Egesten A The human cationic antimicrobial protein (hCAP-18) is expressed in the epithelium of human epididymis, is present in seminal plasma at high concentrations, and is attached to spermatozoa Infect Immun 2000 68 4297 4302 10858248 10.1128/IAI.68.7.4297-4302.2000 Palladino MA Mallonga TA Mishra MS Messenger RNA (mRNA) expression for the antimicrobial peptides beta-defensin-1 and beta-defensin-2 in the male rat reproductive tract: beta-defensin-1 mRNA in initial segment and caput epididymidis is regulated by androgens and not bacterial lipopolysaccharides Biol Reprod 2003 68 509 515 12533413 Zaballos A Villares R Albar JP Martinez AC Marquez G Identification on mouse chromosome 8 of new beta-defensin genes with regionally specific expression in the male reproductive organ J Biol Chem 2004 279 12421 12426 14718547 10.1074/jbc.M307697200 Yamaguchi Y Nagase T Makita R Fukuhara S Tomita T Tominaga T Kurihara H Ouchi Y Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice J Immunol 2002 169 2516 2523 12193721 Com E Bourgeon F Evrard B Ganz T Colleu D Jegou B Pineau C Expression of antimicrobial defensins in the male reproductive tract of rats, mice, and humans Biol Reprod 2003 68 95 104 12493700 Li P Chan HC He B So SC Chung YW Shang Q Zhang YD Zhang YL An antimicrobial peptide gene found in the male reproductive system of rats Science 2001 291 1783 1785 11230693 10.1126/science.1056545 Hamil KG Liu Q Sivashanmugam P Yenugu S Soundararajan R Grossman G Richardson RT Zhang YL O'Rand MG Petrusz P French FS Hall SH Cystatin 11: a new member of the cystatin type 2 family Endocrinology 2002 143 2787 2796 12072414 10.1210/en.143.7.2787 Blankenvoorde MF van't Hof W Walgreen-Weterings E van Steenbergen TJ Brand HS Veerman EC Nieuw Amerongen AV Cystatin and cystatin-derived peptides have antibacterial activity against the pathogen Porphyromonas gingivalis Biol Chem 1998 379 1371 1375 9865612 Jin YZ Bannai S Dacheux F Dacheux JL Okamura N Direct evidence for the secretion of lactoferrin and its binding to sperm in the porcine epididymis Mol Reprod Dev 1997 47 490 496 9211434 10.1002/(SICI)1098-2795(199708)47:4<490::AID-MRD16>3.3.CO;2-A Reddy ES Bhargava PM Seminalplasmin--an antimicrobial protein from bovine seminal plasma which acts in E. coli by specific inhibition of rRNA synthesis Nature 1979 279 725 728 377100 Bourgeon F Evrard B Brillard-Bourdet M Colleu D Jegou B Pineau C Involvement of semenogelin-derived peptides in the antibacterial activity of human seminal plasma Biol Reprod 2004 70 768 774 14613901 Hamil KG Sivashanmugam P Richardson RT Grossman G Ruben SM Mohler JL Petrusz P O'Rand MG French FS Hall SH HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human Endocrinology 2000 141 1245 1253 10698202 10.1210/en.141.3.1245 von Horsten HH Derr P Kirchhoff C Novel antimicrobial peptide of human epididymal duct origin Biol Reprod 2002 67 804 813 12193388 Yenugu S Hamil KG Birse CE Ruben SM French FS Hall SH Antibacterial properties of the sperm-binding proteins and peptides of human epididymis 2 (HE2) family; salt sensitivity, structural dependence and their interaction with outer and cytoplasmic membranes of Escherichia coli Biochem J 2003 372 473 483 12628001 10.1042/BJ20030225 Yenugu S Hamil KG Radhakrishnan Y French FS Hall SH The Androgen Regulated Epididymal Sperm-Binding Protein, ESC42, Is An Antimicrobial Beta-Defensin Endocrinology 2004 145 3165 3173 15033915 10.1210/en.2003-1698 Motizuki M Itoh T Satoh T Yokota S Yamada M Shimamura S Samejima T Tsurugi K Lipid-binding and antimicrobial properties of synthetic peptides of bovine apolipoprotein A-II Biochem J 1999 342 215 221 10432319 10.1042/0264-6021:3420215 Sitaram N Sai KP Singh S Sankaran K Nagaraj R Structure-function relationship studies on the frog skin antimicrobial peptide tigerinin 1: design of analogs with improved activity and their action on clinical bacterial isolates Antimicrob Agents Chemother 2002 46 2279 2283 12069990 10.1128/AAC.46.7.2279-2283.2002 Skerlavaj B Benincasa M Risso A Zanetti M Gennaro R SMAP-29: a potent antibacterial and antifungal peptide from sheep leukocytes FEBS Lett 1999 463 58 62 10601638 10.1016/S0014-5793(99)01600-2 Mangoni ML Papo N Barra D Simmaco M Bozzi A Di Giulio A Rinaldi AC Effects of the antimicrobial peptide temporin L on cell morphology, membrane permeability, and viability of Escherichia coli Biochem J 2004 in press Richards RC O'Neil DB Thibault P Ewart KV Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar) Biochem Biophys Res Commun 2001 284 549 555 11396934 10.1006/bbrc.2001.5020 Yenugu S Richardson RT Sivashanmugam P Wang Z O'Rand MG French FS Hall SH Antimicrobial activity of EPPIN, an androgen regulated sperm bound protein with a whey acidic protein motif. Biol Reprod 2004 in press Errington J Daniel RA Scheffers DJ Cytokinesis in bacteria Microbiol Mol Biol Rev 2003 67 52 65, table of contents 12626683 10.1128/MMBR.67.1.52-65.2003 Ganz T Lehrer RI Antibiotic peptides from higher eukaryotes: biology and applications Mol Med Today 1999 5 292 297 10377520 10.1016/S1357-4310(99)01490-2 Skerlavaj B Romeo D Gennaro R Rapid membrane permeabilization and inhibition of vital functions of gram-negative bacteria by bactenecins Infect Immun 1990 58 3724 3730 2228243 Sharma S Khuller G DNA as the intracellular secondary target for antibacterial action of human neutrophil peptide-I against Mycobacterium tuberculosis H37Ra Curr Microbiol 2001 43 74 76 11375668 10.1007/s002840010263 Patrzykat A Friedrich CL Zhang L Mendoza V Hancock RE Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli Antimicrob Agents Chemother 2002 46 605 614 11850238 10.1128/AAC.46.3.605-614.2002 Sorensen OE Gram L Johnsen AH Andersson E Bangsboll S Tjabringa GS Hiemstra PS Malm J Egesten A Borregaard N Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina J Biol Chem 2003 278 28540 28546 12759353 10.1074/jbc.M301608200 Yudin AI Tollner TL Li MW Treece CA Overstreet JW Cherr GN ESP13.2, a member of the beta-defensin family, is a macaque sperm surface-coating protein involved in the capacitation process Biol Reprod 2003 69 1118 1128 12773404 Zhou CX Zhang YL Xiao L Zheng M Leung KM Chan MY Lo PS Tsang LL Wong HY Ho LS Chung YW Chan HC An epididymis-specific beta-defensin is important for the initiation of sperm maturation Nat Cell Biol 2004 6 458 464 15122269 10.1038/ncb1127

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==== Front Reprod Biol EndocrinolReproductive biology and endocrinology : RB&E1477-7827BioMed Central London 1477-7827-2-631534506010.1186/1477-7827-2-63ResearchImmature rats show ovulatory defects similar to those in adult rats lacking prostaglandin and progesterone actions Gaytan María [email protected] Carmen [email protected] Concepcion [email protected] Marcelino [email protected] Jose E [email protected] Francisco [email protected] Department of Cell Biology, Physiology and Immunology, School of Medicine, University of Cordoba, Spain2 Department of Pathology, School of Medicine, University of Cordoba, Spain2004 3 9 2004 2 63 63 2 7 2004 3 9 2004 Copyright © 2004 Gaytan et al; licensee BioMed Central Ltd.2004Gaytan et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gonadotropin-primed immature rats (GPIR) constitute a widely used model for the study of ovulation. Although the equivalence between the ovulatory process in immature and adult rats is generally assumed, the morphological and functional characteristics of ovulation in immature rats have been scarcely considered. We describe herein the morphological aspects of the ovulatory process in GPIR and their response to classical ovulation inhibitors, such as the inhibitor of prostaglandin (PG) synthesis indomethacin (INDO) and a progesterone (P) receptor (PR) antagonist (RU486). Immature Wistar rats were primed with equine chorionic gonadotropin (eCG) at 21, 23 or 25 days of age, injected with human chorionic gonadotropin (hCG) 48 h later, and sacrificed 16 h after hCG treatment, to assess follicle rupture and ovulation. Surprisingly, GPIR showed age-related ovulatory defects close similar to those in adult rats lacking P and PG actions. Rats primed with eCG at 21 or 23 days of age showed abnormally ruptured corpora lutea in which the cumulus-oocyte complex (COC) was trapped or had been released to the ovarian interstitum, invading the ovarian stroma and blood and lymphatic vessels. Supplementation of immature rats with exogenous P and/or PG of the E series did not significantly inhibit abnormal follicle rupture. Otherwise, ovulatory defects were practically absent in rats primed with eCG at 25 days of age. GPIR treated with INDO showed the same ovulatory alterations than vehicle-treated ones, although affecting to a higher proportion of follicles. Blocking P actions with RU486 increased the number of COC trapped inside corpora lutea and decreased ovulation. The presence of ovulatory defects in GPIR, suggests that the capacity of the immature ovary to undergo the coordinate changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age. ==== Body Introduction Ovulation, the release of mature oocytes from the ovary, requires proteolytic degradation of the follicle wall, as well as the overlying ovarian tissues. This happens through the expression of a series of critical genes, triggered in a precise temporal and spatial pattern by the preovulatory LH surge [1,2]. It is worthy to note that, for successful ovulation, follicle rupture has to occur just at the site of the follicle wall facing the ovarian surface, thus allowing release of the cumulus-oocyte complex (COC) to the periovarian space, while preventing proteolytic damage of the perifollicular tissues at the basolateral follicle sides. A large amount of information on the ovulatory process was accumulated during the last century (reviewed in [1-5]), and the involvement of crucial genes such as those encoding cyclooxygenase-2 (COX-2), and progesterone receptor (PR) has been clearly established. However, the mechanisms underlying the spatial targeting of the follicle rupture remain poorly understood. Although mechanical factors are likely involved in stigma formation and rupture [6], the mechanisms responsible for the specific location of proteolytic breakdown of the theca layers and perifollicular connective tissue at the apex of the follicle are not known. In recent studies [7-9] we have proposed that both prostaglandins (PG) and progesterone (P), classically recognized as essential ovulatory factors [1,2], play complementary roles in the spatial targeting of follicle rupture. This was supported by detailed morphological studies in cycling rats treated with indomethacin (INDO), a strong inhibitor of PG synthesis, and RU486 (a PR antagonist), showing antiovulatory effects [1,2,10-12]. Gonadotropin-primed immature rats (GPIR) constitute a useful model for the study of ovulation. The administration of a single dose of equine chorionic gonadotropin (eCG) to immature animals induces the growth of abundant follicles, that reach preovulatory size in two days. Ovulation is then triggered by a single dose of human chorionic gonadotropin (hCG), thus providing a large number of synchronized ovulatory follicles [13-25]. An additional advantage of this model is the absence of regressing corpora lutea of previous cycles. This is relevant because structural luteolysis, that is temporally coincident with ovulation in cycling rats, also involves tissue remodeling and proteolytic degradation of the extracellular matrix [5]. For these reasons, GPIR (ranging from 21 to 28 days of age, at the time of eCG treatment [13-25]), have been widely used in studies focused on the ovulatory process, and a large amount of the information in this topic is derived from studies in immature rats. However, it should be kept in mind that GPIR constitute a non-physiological model and the possible immaturity of the pathways leading to ovulation cannot be ruled out. In order to examine further the role of PG and P in follicle rupture and ovulation, we performed detailed morphological analysis of the ovulatory process in inmature rats primed with gonadotropins at different ages. Surprisingly, these animals showed age-related alterations of the follicle rupture similar to those of adult rats lacking PG and P actions. We report herein the morphological alterations of follicle rupture and ovulation in GPIR and the effects of treatment with P and/or PG of the E series, as well as the response of GPIR to PG synthesis inhibition with INDO and to a PR antagonist (RU486), whose inhibitory effects in ovulation are clearly established [1,2,8,13,14]. Materials and methods Animals and drugs Wistar female rats bred in the vivarium of the University of Cordoba were used. The day the litters were born was considered as day 0. Litter size was adjusted to 8 pups. The animals were maintained under controlled conditions of light (14L:10D; lights on 0500-1900) and temperature (22°C). The animals had free access to pelleted food and tap water. Experimental procedures were approved by the Cordoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union guidelines for care and use of experimental animals. Indomethacin (INDO), Progesterone (P), equine chorionic gonadotropin (eCG), human chorionic gonadotropin (hCG) and prostaglandins were purchased from Sigma.(St Louis, MO). The progesterone antagonist RU486 was obtained from Exlegin (Paris, France). Gonadotropin-priming at different ages Wistar immature rats were injected sc with 10 IU of eCG at 1700 h at 21, 23 or 25 days of age, and 48 h later were injected sc with 10 IU of hCG, a frequently used schedule [23,26,27]. Body weights were in the recommended range [23,26] (46.0 ± 0.34, 57.0 ± 1.30 and 61.8 ± 1.4 g, mean ± SEM for n = 5). The animals (5 per group) were sacrificed at 0900 h on the day following hCG injection (i.e. at 24, 26 and 28 days of age). The ovaries, including the ovarian bursa, oviducts and periovarian fat pad, were fixed in Bouin-Hollande's fluid for 24 h and processed for paraffin embedding. Gonadotropin-primed rats treated with prostaglandins and/or progesterone Immature rats were primed with eCG at 21 days of age and with hCG 48 h later as described above. In addition, these animals were injected with 100 μg of PGE1 or PGE2, 1 mg of P or 1 mg of P plus 100 μg of PG E1 or PGE2 or vehicles (70% ethanol in saline) at 1730 h (30 min after hCG treatment). These dosages were effective, in the same body weight basis, in previous studies in adult rats [9]. The animals (5 per group) were killed at 0900 h on the day after hCG treatment (i.e. at 24 days of age) and the ovaries were processed as described above. Gonadotropin.primed rats treated with indomethacin or RU486 Immature rats were primed with eCG at 23 days of age and with hCG 48 h later as described in previous experiments. Indomethacin-treated rats received a sc injection of 0.5 mg of INDO or vehicle (olive oil) at 1200 h at 25 days of age. RU486-treated rats were injected sc with 0.5 mg of RU486 or vehicle (olive oil) at 0900 h at 24 and 25 days of age.The animals (5 per group) were killed at 0900 h on the day following hCG injection (i.e. at 26 days of age) and the ovaries were processed as described above. Histological analysis of follicle rupture and ovulation The right ovaries were serially sectioned (6 μm) and stained with hematoxylin and eosin. All sections were examined under the microscope. The total number of cumulus-oocyte complexes (COCs) per ovary were counted. The number of COCs trapped inside the corpus luteum, released to the ovarian interstitium, retained in the bursal cavity or found in the oviducts, was recorded. COCs released to the ovarian interstitium were clearly recognizable by the presence of the oocyte in metaphase II and the first polar body, dispersed cumulus and follicular fluid. The total number of COCs per ovary matches the total number of corpora lutea (unruptured or not). In addition to absolute values, the number of COCs in each location was expressed as the percentage with respect to the total number of COCs (or corpora lutea) per ovary, to assess ovulatory efficiency, avoiding variability in the total numbers of corpora lutea among the different groups. Unruptured follicles measuring less than 575 μm in diameter, showing signs of atresia, such as irregularities of the granulosa cell layer, presence of apoptotic granulosa cells, as well as lack of dispersion of the cumulus or resumption of meiosis, were not considered. Statistical analysis was performed by ANOVA followed by the Student-Newman-Keuls method for multiple comparison among means. Significance was considered at the 0.05 level. Results Ovulatory process in immature rats primed at different ages Absolute numbers of COCs, that match the numbers of corpora lutea per ovary, are presented in Table 1, whereas relative data (the proportion of COCs found in each specific location) are shown in Fig. 6. On the day following hCG treatment, the total number of COCs per ovary was equivalent for the different age-groups (Table 1). Immature rats primed with eCG at 21 or 23 days of age showed frequent ovulatory defects (see Figs 1,2,3,4,5 for representative micrographs and Fig. 6. for quantitative data). The COC was trapped in about 60% and 40% of the luteinized follicles in rats primed with eCG at 21 and 23 days of age respectively (Figs. 1A, 2, 6). However, many luteinized follicles containing the COC showed rupture of the theca layers and release of follicular fluid to the ovarian interstitium (Fig. 1A). Furthermore, 6–10% of the COCs were released to the ovarian interstitum (Figs. 1B, 2, 3, 6). In these cases, degradation of the ovarian stroma with invasion of the blood and lymphatic vessels and formation of emboli containing the COC and follicular fluid (Figs 1C,1D,1E,1F, 2, 3) were observed. In some animals, massive embolism of the ovarian vein at the ovarian hilus with follicular fluid and COCs (Fig. 3) caused ovarian hyperemia and vascular congestion. In addition, proteolytic degradation of the ovarian bursa by follicular fluid and granulosa cells, with invasion of the periovarian fat pad (Fig. 1G,1H) and even release of the COC to the peritoneal cavity (Fig. 4), was also observed. A characteristic feature of some corpora lutea ruptured at the apex, was the presence of an unruptured ovarian surface epithelium, and the COC was trapped in lacunae of follicular fluid and blood in the tunica albuginea (Fig. 5). Variable numbers of COCs (from 0 to 25%) were located in the bursal cavity (Fig. 6). From 33% to 60% of COCs were found in the oviducts, in rats primed with eCG at 21 and 23 days of age respectively. Otherwise, in immature rats primed with eCG at 25 days of age, the vast majority of COCs were found in the oviducts and abnormally ruptured corpora lutea (with trapped COC) were only occasionally found. Table 1 Number of COCs per ovary in control rats primed with eCG at different ages. Age (days) at eCG treatment Total COCs COCs located in Corpus luteum Interstitium Ovarian Bursa Oviduct 21 42.5 ± 8.72 22.5 ± 4.32 3.17 ± 1.47 0.67 ± 0.42 16.2 ± 6.93 23 58.4 ± 4.74 18.8 ± 0.70 3.20 ± 1.74 2.40 ± 0.51 34.0 ± 6.54 25 38.6 ± 3.76 0.6 ± 0.60a,b -- -- 37.4 ± 3.20 Values are the mean ± SEM for n = 5. ANOVA and Student-Newman-Keuls multiple range test. a p < 0.05 vs 21 days. b p < 0.05 vs 23 days Figure 1 Representative micrographs from the ovary of immature rats primed with eCG at 21 (A-D, G, H) or 23 (E, F) days of age, stained with hematoxylin and eosin. A, luteinized follicle showing trapped COC and release of follicular fluid (FF) to the ovarian interstitium. The rupture of the theca layers are indicated by arrows. B, COC released to the ovarian interstitium, in a lacunae of follicular fluid (FF). Clusters of granulosa cells are indicated by open arrows. C, D, COCs in the lymphatic (A) or blood (D) vessels at the ovarian hilus (OH). E, F, COC inside a blood vessel located in the periovarian fat pad (PFP) near the ovarian hilus (OH). The framed area is shown at higher magnification in F showing rupture of the blood vessel and escape of red blood cells (arrows). G, H, Non-consecutive serial sections showing a COC released to the bursal cavity (BC), adhered to the ovarian bursa. Degradation of the ovarian bursa by follicular fluid and granulosa cells (open arrows) and invasion (arrows) of the periovarian fat pad (PFP) can be observed. Figure 2 Micrograph of the ovary of a rat primed with eCG at 21 days of age, stained with hematoxylin and eosin. Several COCs trapped inside corpora lutea, showing dispersion of the cumulus, and interstitial COCs inside a blood vessel. Figure 3 Non-consecutive serial sections of the ovary of a rat primed with eCG at 21 days of age and stained with hematoxylin and eosin. The ovarian vein is embolized with folicular fluid and two COCs (in A and B). A COC released to the ovarian hilus can be also observed in A. The ovarian surface is indicated by empty arrows in the inset. Figure 4 Micrographs from non-consecutive serial sections from the ovary of an immature rat primed with eCG at 21 days of age. The ovarian bursa (OB) has been degraded (A) allowing release of the COC to the peritoneal cavity (B) Clusters of granulosa cells (arrows) can be observed free or attached to the ovarian bursa. Hematoxylin and eosin. Figure 5 Representative micrographs from the ovary of immature rats primed with eCG at 21 (A) or 23 (B) days of age, showing luteinized follicles ruptured at the apex. The ovarian surface epithelium (OSE, arrows in A and B), and its basement membrane (arrowheads in B) remain intact, whereas the underlying ovarian tissue has been degraded (asterisk in A). The COC is retained under the ovarian surface. OB, ovarian bursa. Hematoxylin and eosin. Figure 6 Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in immature rats primed with eCG at 21, 23 or 25 days of age and ovulating at 24, 26 or 28 days of age respectively. See Table 1 for absolute counts. Different superscripts mean significant (p < 0.05) differences. Effects of P and/or PGE treatment The total number of COCs per ovary was equivalent in rats primed with eCG at 21 days of age and treated with vehicles, P, PGE1, PGE2, P plus PGE1 or P plus PGE2 (Table 2). Neither P, PG or combined P plus PG treatment, prevented ovulatory defects and these animals showed the same morphological alterations as gonadotropin and vehicle-treated rats. However, degradation of the interstitial tissue and embolism of blood vessels with follicular fluid and granulosa cells were very scarce in prostaglandin-treated animals. Although the number of effectively ovulated oocytes were apparently increased in prostaglandin-treated rats, differences were not large enough to be statistically significant (Fig. 7). Table 2 Number of COCs per ovary in rats primed with eCG at 21 days of ages. Treatment Total COCs COCs located in Corpus luteum Interstitium Ovarian Bursa Oviduct Vehicles 35.4 ± 3.52 17.2 ± 1.47 2.6 ± 0.85 0.6 ± 0.24 15.0 ± 2.72 PGE1 33.2 ± 4.20 11.0 ± 2.42 2.3 ± 1.02 1.2 ± 0.58 19.0 ± 3.40 PGE2 33.2 ± 1.62 10.2 ± 2.80 0.6 ± 0.39 3.6 ± 2.59 18.8 ± 3.38 P4 45.6 ± 7.77 17.2 ± 2.97 3.4 ± 1.06 5.8 ± 3.10 19.2 ± 5.45 P4 + PGE1 34.6 ± 5.17 14.6 ± 3.37 0.6 ± 0.39 0.8 ± 0.37 18.6 ± 2.82 P4 + PGE2 35.2 ± 6.85 15.6 ± 3.10 0.6 ± 0.39 5.7 ± 2.85 14.2 ± 1.63 Values are the mean ± SEM for n= 5. Figure 7 Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in rats primed with e CG at 21 days of age and injected two days later with vehicles (Veh), prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), progesterone (P) or progesterone plus PGE1 or PGE2. See Table 2 for absolute counts. No significant differences were found. Effects of INDO or RU486 treatments The total number of COCs per ovary was equivalent in rats treated with vehicle, INDO or RU486 (Table 3). Immature rats treated with INDO during the preovulatory period showed the same morphological alterations of the ovulatory process as gonadotropin and vehicle-treated rats, although a higher proportion of follicles were affected (Fig. 8). A significantly (p < 0.05) higher number of COCs were released to the ovarian interstitium, whereas the number of COCs found in the oviducts was significantly (p < 0.05) decreased. Rats treated with RU486 showed increased numbers of COCs retained inside the follicle (Fig. 8), whereas the number of COCs released to the interstitium or effectively ovulated were significantly decreased. Most of the luteinized follicles in which the COC was trapped, did not show evident rupture of the theca layers. The numbers of COC trapped in the bursal cavity was significantly increased (Table 3 and Fig. 8). Table 3 Number of COCs per ovary in rats primed with eCG at 23 days of ages. Treatment Total COCs COCs located in Corpus luteum Interstitium Ovarian Bursa Oviduct Vehicle 39.6 ± 1.60 12.8 ± 1.0 2.6 ± 1.73 1.0 ± 0.62 23.2 ± 1.53 INDO 34.4 ± 3.76 19.2 ± 2.55 10.0 ± 1.35a 0.4 ± 0.39 5.0 ± 0.05a RU486 41.8 ± 3.86 22.8 ± 4.80 0.6 ± 0.39b 3.2 ± 0.57a,b 15.2 ± 3.10a,b Values are the mean ± SEM for n = 5. ANOVA and Student-Newman-Keuls multiple range test. a p < 0.05 vs vehicle, b p < 0.05 vs INDO. Figure 8 Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in rats primed with eCG at 23 days of age and treated with indomethacin (INDO) or RU486. see Table 3 for absolute counts. Different superscripts mean significant (p < 0.05) differences. Discussion Gonadotropin-primed immature rats (GPIR) is a popular model for the study of ovulation. In this model, eCG stimulates the growth of a large cohort of follicles, that reach preovulatory size in about 48 h and are then induced to ovulate by hCG [1,2,28]. Early data indicate that the ovulation rate in immature rats was age-dependent, and that the number of ova found in the oviducts increases, although not linearly, with age [29,30]. However, these early studies do not provide information on the number of ova that were not effectively ovulated, and the marked variability in the rate of ovulation at different ages could be due to differences in the number of recruited growing follicles, in the responsiveness of preovulatory follicles to the ovulatory stimulus or both. In the present study, we evaluated the ovulatory process in immature rats primed with gonadotropins during the 21–25 day-old period, commonly used in studies focused on ovulation [13,17,18,20-23,25,26]. In agreement with previous data [29,30], the proportion of COCs found in the oviducts (i.e. effectively ovulated) increases in parallel to age. Detailed morphological evaluation indicated that ovulation was unsuccessful in 30–60% of preovulatory follicles in rats primed with eCG before 25 days of age. The nearly normal ovulatory process in immature rats primed with eCG at 25 days of age indicates that ovulatory defects were not due to gonadotropin treatment itself, but to ovarian immaturity at earlier ages. The ovulation failure described herein (in rats primed with eCG at 21 or 23 days of age) was not due to decreased recruitment of growing follicles, as indicated by the equivalent numbers of follicles that reach preovulatory size at all ages tested. The data of this study strongly suggest that the relative ovulatory incompetence of young immature rats was due to a defective capacity of preovulatory follicles to undergo the coordinate network of interactions that leads, in reponse to hCG, to COC release to the periovarian space. Defective ovulation in GPIR was related to abnormal follicle rupture. Whereas some LH-driven morphological changes in preovulatory follicles, such as cumulus expansion, resumption of the meiotic process and initial luteinization were present and showed normal features in GPIR, aberrant follicle ruptures were frequently observed. This was indicated by the presence of COCs released to the ovarian interstitium, and of corpora lutea showing rupture of the theca layers at any site of the follicle wall with release of follicular fluid to the ovarian stroma. The presence of follicle ruptures at the basolateral follicle sides suggests that the mechanisms underlying the spatial targeting of follicle rupture at the apex are not fully established in immature rats primed with eCG before 25 days of age. Almost identical ovulatory defects have been previously reported in INDO-treated adult cycling rats that also show abnormally ruptured follicles and proteolytic degradation of perifollicular tissues [7-9]. Furthermore, INDO-treated GPIR in the present study showed similar morphological alterations of the ovulatory process as vehicle-treated GPIR, even though the drug increased the number of affected follicles. The similarity of the ovulatory alterations found in GPIR and INDO-treated adult rats raises the question of whether the COX-2-prostaglandin pathway is fully established or not in GPIR. In this context, treatment of GPIR with PG of the E series was carried out in order to analyse whether ovulatory defects in GPIR were due to defective PG synthesis. Treatment with prostaglandin E resulted in a decrease in the invasion of the ovarian stroma and blood vessels by follicular fluid and granulosa cells, but ovulation was not improved significantly. In contrast, supplementation with exogenous PG of the E series inhibits abnormal follilce rupture and restores ovulation in INDO-treated adult rats [2,8,31]. This suggests that the possible immaturity of the COX-2-prostaglandin pathway in GPIR would be beyond prostaglandin synthesis, in agreement with previous studies reporting normal PG generation in GPIR in response to hCG treatment [28]. Otherwise, recent studies in COX-2 defective mice [32] indicate that the COX-2-PG pathway is not fully established in immature rodents. It has been clearly established that activation of PR plays a crucial role in ovulation [10,11,33-37]. Accordingly, adult cycling rats treated with PR antagonists [11] or inhibitors of the P synthesis [34] during the preovulatory period showed almost complete inhibition of follicular rupture [9,11,34]. Noteworthy, the response of GPIR to PR blockage (showing incomplete inhibition of follicle rupture) was similar to that of adult rats lacking both P and PG actions. In this sense, combined treatment of adult rats with RU486 and INDO resulted in follicle rupture in about 25% of preovulatory follicles [9]. Furthermore, some morphological features of the ovulatory process in GPIR, such as the persistence of the ovarian surface epithelium in spite of the degradation of the underlying tissues, was also a characteristic feature of adult rats treated with both INDO and RU486 [9]. However, exogenous P supplementation did not restore ovulation in GPIR, that suggest a defective response of preovulatory follicles to the ovulatory LH-dependent P surge. RU486-treated GPIR showed a significant increase in the number of COCs retained into the bursal cavity. Alterations in the transport of COCs to the oviduct has been previously described in adult rats treated with a PR antagonist [11], although the existence of a role for P in the COC transport to the oviduct cannot be ascertained from the present data. The end-point of the complex interaction network leading to follicle rupture and COC release, is the expression/activation of proteolytic enzymes, reponsible for extracellular matrix breakdown [24,38,39]. Proteolytic activity has to be closely modulated by protease inhibitors to prevent damage to perifollicular tissues. However, the characterization and regulation of ovarian proteolytic inhibitors has not been completely elucidated. The presence in GPIR of degradation of the ovarian stroma, invasion of blood and lymphatic vessels in abnormally ruptured follicles, strongly suggest that defective ovulation in GPIR was not due to decreased proteolytic activity but due to untargeted proteolysis. This was particularly suggested by the degradation of the ovarian bursa and invasion of the periovarian fat pad by cumulus cells and follicular fluid released at the follicle apex (see Figs. 1G,1H, 3 and 4), permitting, in some cases, escape of the COC to the peritoneal cavity. In this sense, the use of very young immature rats to characterize proteolytic activity regulation during ovulation could provide conflicting data. In this study, we used Wistar rats. The possible existence of slight differences in the age of ovarian maturation among different rat strains cannot be ruled out. In conclusion, the presence of aberrant ovulations in very young GPIR, closely resembling those in adult rats lacking P and PG actions, strongly suggests that the capacity of the immature ovary to undergo changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age. This should be taken into account when using the GPIR model for studies focused on ovulation. Acknowledgements This work has been subsidized by Grant BFI2002-00485 from the DGI, Spain. The authors are very grateful to J Molina, P Cano and E Tarradas for their technical assistance. ==== Refs Tsafriri A Chun SY Reich R Adashi EY, Leung PCK Follicular rupture and ovulation The Ovary 1993 New York: Raven Press 228 243 Espey LL Lipner H Knobil E, Neill JD Ovulation The physiology of Reproduction 1994 1 2 New York: Raven Press 725 780 Brännström M Janson PO Hillier SG The biochemistry of ovulation Ovarian Endocrinology 1991 Oxford: Blackwell Scientific Publications 132 166 Robker RL Russell DL Yoshioka S Sharma SC Lydon JP O'Malley BW Espey LL Richards JS Ovulation: a multi-gene, multi-step process Steroids 2000 65 559 570 11108860 10.1016/S0039-128X(00)00114-8 Ny T Wahlberg P Brändström IJM Matrix remodeling in the ovary: regulation and functional role of the plasminogen activator and matrix metalloproteinase systems Mol Cell Endocrinol 2002 187 29 38 11988309 10.1016/S0303-7207(01)00711-0 Rodbard D Mechanics of ovulation J Clin Endocrinol Metab 1968 28 849 861 5656439 Gaytán F Tarradas E Morales C Bellido C Sánchez-Criado JE Morphological evidence for uncontrolled proteolytic activity during the ovulatory process in indomethacin-trated rats Reproduction 2002 123 639 649 12006092 10.1530/rep.0.1230639 Gaytán F Tarradas E Bellido C Morales C Sánchez-Criado JE Prostaglandin E1 inhibits abnormal follicle rupture and restores ovulation in indomethacin-treated rats Biol Reprod 2002 67 1140 1147 12297529 Gaytán F Bellido C Gaytán M Morales C Sánchez-Criado JE Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat Biol Reprod 2003 69 99 105 12606368 Van der Schoot P Bakker GH Kljin JG of the progesterone antagonist RU486 on ovarian activity in the rat Endocrinology 1987 121 1375 1382 3653032 Sánchez-Criado JE Bellido C Galiot F López FJ Gaytán F A posible dual mechanism of the anovulatory action of antiprogesterone RU486 in the rat Biol Reprod 1990 42 877 886 2116924 Reese J Zhao X Ma W Brown N Maziasz TJ Dey SK Comparative analysis of pharmacologic and/or genetic disruption of cyclooxygenase-1 and cyclooxygenase-2 function in female reproduction in mice Endocrinology 2001 142 3198 3206 11416042 10.1210/en.142.7.3198 Mori T Kohda H Kinoshita Y Ezaki Y Morimoto N Nishimura T Inhibition by indomethacin of ovulation induced by human chorionic gonadotrophin in immature rats primed with pregnant mare serum gonadotrophin J Endocrinol 1980 84 333 341 7391712 Rao IM Mahesh VB Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat Biol Reprod 1986 35 1154 1161 3103699 Butler TA Zhu C Mueller RA Fuller GC Lemaire WJ Woessner JF Inhibition of ovulation in the perfused rat ovary by the synthetic collagenase inhibitor SC 44463 Biol Reprod 1991 44 1183 1188 1714777 Zhu C Woessner JF Jr A tissue inhibitor of metalloproteinases and α-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown Biol Reprod 1991 45 334 342 1723897 Chun SY Popliker M Reich R Tsafriri A Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary Biol Reprod 1992 47 245 253 1327205 Iwamasa J Shibata S Tanaka N Matsuura K Okamura H The relationship between ovarian progesterone and proteolytic enzyme activity during ovulation in the gonadotropin-treated immature rat Biol Reprod 1992 46 309 313 1536908 Mann JS Kindy MS Hyde JF Clark MR Curry TE Jr Role of protein synthesis, prostaglandins, and estrogen in rat ovarian metalloproteinase inhibitor production Biol Reprod 1993 48 1006 1013 8481463 Natraj U Richards J Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles Endocrinology 1993 133 761 769 8344215 10.1210/en.133.2.761 Liu K Wahlberg P Ny T Coordinated and cell-specific regulation of memebrane type matrix metalloproteinase 1 (MT1-MMP) and its substrate matrix metalloproteinase 2 (MMP-2) by physiological signals during follicular development and ovulation Endocrinology 1998 139 4735 4738 9794486 10.1210/en.139.11.4735 Cooke RG IIINothnick WB Komar C Burns P Curry TE Jr Collagenase and gelatinase messenger ribonucleic acid expression and activity during follicular development in the rat ovary Biol Reprod 1999 61 1309 1316 10529279 Espey LL Yoshioka S Russel D Ujioka T Vladu B Skelsey M Fujii S Okamura H Richards JS Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature rat Biol Reprod 2000 62 390 397 10642578 Curry TE JrSong L Wheeler SE Cellular localization of gelatinases and tisssue inhibitors of metalloproteinases during follicular growth, ovulation, and early luteal formation in the rat Biol Reprod 2001 65 855 865 11514351 Simpson KS Komar CM Curry TE Jr Localization and expression of tissue inhibior of metalloproteinase-4 in the immature gonadotropin-stimulated and adult rat ovary Biol Reprod 2003 68 214 221 12493716 Tanaka N Espey LL Stacy S Okamura H Epostane and indomethacin actions on ovarian kalikrein and plasminogen activator activities during ovulation in the gonadotropin-primed immature rat Biol Reprod 1992 46 665 670 1576264 Yoshioka S Fujii S Richards JS Espey LL Gonadotropin-induced expression of pancreatitis-associated protein-III mRNA in the rat ovary at the time of ovulation J Endocrinol 2002 174 485 492 12208669 Espey LL Norris C Forman J Siler-Khodr T Effect of indomethacin, cycloheximide, and aminoglutethimide on ovarian steroid and prostanoid levels during ovulation in the gonadotropin-primed immature rat Prostaglandins 1989 38 531 539 2602561 10.1016/0090-6980(89)90147-0 Zarrow MX Wilson ED The influence of age on superovulation in the immature rat and mouse Endocrinology 1961 69 851 855 13787954 Zarrow MX Quinn DL Superovulation in the immature rat following treatment with SG-induced ovulation J Endocrinol 1963 26 181 188 14003280 Sogn JH Curry TEJr Brännström M LeMaire WJ Koos RD Papkoff H Janson PO Inhibition of follicle-stimulating hormone-induced ovulation by indomethacin in the perfused rat ovary Biol Reprod 1987 36 536 542 3109504 Matsumoto H Ma W Smalley W Trzaskos J Breyer RM Dey SK Diversification of cyclooxygenase-2-derived prostaglandins in ovulation and implantation Biol Reprod 2000 64 1557 1565 11319164 Mori T Suzuku A Nishimura T Kambegawa A Inhibition of ovulation in immature rats by antiprogesterone antiserum J Endocrinol 1977 73 185 186 858983 Snyder B Beecham G Schane H Inhibition of ovulation in rats with epostane, an inhibitor of 3β-hydroxysteroid dehydrogenase Proc Soc Exp Biol Med 1984 176 238 242 6587400 Hibbert ML Stouffer RL Wolf DP Zelinski-Wooten MB Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates Proc Natl Acad Sci USA 1996 93 1897 1901 8700855 10.1073/pnas.93.5.1897 Uilenbroek JTJ Hormone concentration and ovulatory response in rats treated with antiprogestagens J Endocrinol 1991 129 423 429 2066697 Pall M Mikuni M Mitsube K Brännström M Time-dependent ovulation inhibition of a selective progesterone-receptor antagonist (Org 31710) and effects on ovulatory mediators in the in vitro perfused rat ovary Biol Reprod 2000 63 1642 1647 11090431 Reich R Tsafriri A Mechanic GL The involvement of collagenase in ovulation in the rat Endocrinology 1985 116 522 527 2981665 Woessner JF JrButler T LeMaire WJ Morioka N Mukaida T Zhu C Tsafriri A, Dekel N The role of collagenase in ovulation in the rat Follicular Development and Ovulation Regulation 1989 Serono Symposia: Raven Press 169 178

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==== Front J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-1-171532769010.1186/1742-2094-1-17ResearchHuman oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAs Hosokawa Masato [email protected] Andis [email protected] Patrick L [email protected] Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada2004 24 8 2004 1 17 17 20 5 2004 24 8 2004 Copyright © 2004 Hosokawa et al; licensee BioMed Central Ltd.2004Hosokawa et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis. This vulnerability may be related to a deficiency in their ability to express complement regulatory proteins. Methods This study compared the expression level of complement inhibitor mRNAs by human oligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR. Results Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expression was dramatically lower in oligodendroglial cells compared with astrocytes and microglia. The mRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was also significantly lower than for other cell types. Conclusion The lower mRNA expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system. ==== Body Background Resident brain cells including oligodendrocytes [1,2], astrocytes, astrocytomas, microglia, glioblastomas [3-14], neurons [15,16], neuroblastomas [17,18] and endothelial cells [19,20] express mRNAs for complement proteins. Although the role of complement expression by these cells remains unclear, local complement activation in the central nervous system (CNS) might damage these cells and contribute to the pathology in several inflammatory and neurodegenerative diseases including multiple sclerosis, Alzheimer's disease and progressive supranuclear palsy. For self-protection, resident brain cells also express complement inhibitors, such as membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligodendroglial cell line produces MCP, DAF, CD59, C1-inh and S-protein, but not complement receptor 1 (CR1) [1]. Human oligodendrocytes have been reported to express CD59 [21] and DAF, but not MCP, CR1, homologous restriction factor (HRF: C8 bp) or clusterin [22]. Astrocytes [23], neurons and Schwann cells have been reported to express CD59 [24] and neuroblastoma cell lines C1-inh [18]. Astrocytoma cell lines have been reported to express MCP, DAF, and CD59 [25,26]. In this study, the expression level of mRNAs for various complement inhibitors by human oligodendrocytes, astrocytes and microglia were compared by semi-quantitative PCR. We show that oligodendrocytes express extremely low levels of mRNA for C1-inh and significantly lower levels of mRNA for MCP compared with astrocytes and microglia. The expression level of mRNAs for CD59 and DAF showed no significant differences between the three cell types. Methods Cell culture: microglial- and astrocyte-enriched cultures Human microglial and astrocytic cells were isolated from surgically resected temporal lobe tissues. We thank Dr. J. Maguire, Department of Pathology and Laboratory Medicine, Vancouver General Hospital for providing the surgical specimens. Isolation protocols described by De Groot et al. [27,28] were used with minor modifications. Tissues were placed in a sterile Petri dish, rinsed with Hank's balanced salt solution, and visible blood vessels were removed. After washing tissues two more times with Hank's balanced salt solution, tissues were chopped into small (<2 mm3) pieces with a sterile scalpel. The fragments were transferred into a 50 ml centrifuge tube containing 10 ml of 0.25% trypsin solution (Gibco-BRL, Life Technologies, Burlington, ON, Canada), and incubated at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d'Urfé, PQ, Canada) was added to reach a final concentration of 50 μg/ml. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F12 ham (DMEM-F12; Sigma-Aldrich, Oakville, ON, Canada) with 10% fetal bovine serum (FBS; Gibco-BRL, Life Technologies), and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 × g for 10 min, the cell pellet was resuspended in serum containing medium, triturated several times, and passed through a 100 μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged once more (275 × g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin (50 μg/ml, from Sigma), and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO2, 95% air atmosphere at 37°C for 2 hr in order to achieve adherence of microglial cells. Non-adherent cells with myelin debris were removed from these microglia-enriched cultures and transferred into poly-L-lysine coated 10 cm tissue culture plates in order to achieve adherence of astrocytes. Plates were incubated for 48 hr, after which the culture medium containing myelin debris and non-adherent cells was removed and used to prepare oligodendroglial cell cultures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before their mRNAs were extracted. Immunostaining with antibodies against CD68 (Dako, Mississauga, ON, Canada) which stains microglia as well as macrophages, and glial fibrillary acidic protein (GFAP, Dako), which is a marker of astrocytes, showed that the microglia-enriched cultures contained 93.5 ± 3.6 % (N = 4) microglial cells, while astrocyte-enriched cultures contained 85.7 ± 3.4 % (N = 4) astrocytes. Cell culture: oligodendroglial cells These were prepared as described before [2]. Briefly, cell culture media containing myelin debris and non-adherent cells that were removed from astrocyte-enriched cultures were used to extract oligodendroglial cells. The non-adherent cells were collected by centrifugation at 275 × g for 10 min and replated onto uncoated 10 cm tissue culture plates for another 24 hr. Subsequently, the cell culture medium containing floating cells was transferred to 50 ml tubes and Lymphoprep solution (Axis-Shield, Oslo, Norway) used to reduce the amount of contaminating myelin debris. For this purpose, 10 ml of Lymphoprep solution was carefully placed under the oligodendrocyte cell suspension and the density gradient was centrifuged at 325 × g for 10 min. The interphase was collected and transferred to a 50 ml centrifuge tube. Fresh culture medium was added and the suspension was centrifuged at 275 × g for 7 min. The cell pellet was resuspended and the oligodendrocyte cultures seeded onto 60 mm plastic culture dishes. Immunostaining with anti-O4 antibody (Chemicon International, Temecula, CA), which is a marker of oligodendrocytes, showed that the oligodendrocytes-enriched cultures contained 95.3 ± 4.4 % (N = 4) oligodendrocytes. RNA isolation and cDNA synthesis by reverse transcription Total RNA from oligodendroglial cells, microglia, and astrocytes were isolated by the acid guanidium thiocyanate-phenol-chloroform method. Two μg of the RNA was then used to prepare cDNA. RNA was treated with 10 U of DNase I (Gibco BRL, Life Technologies) for 60 min at 37°C in 25 μl of 1 × reverse transcriptase buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2) containing 40 U of RNase inhibitor (Pharmacia Biotech) and 1 mM dithiothreitol (DTT), following by incubation at 85°C for 5 min to inactivate the enzyme. Reverse transcription was performed at 42°C for 90 min in 50 μl of the following mixture: 1 × reverse transcriptase buffer containing 2 μg of RNA, 5 mM DTT, 0.2 μg random hexamer primers (Pharmacia Biotech), 1 mM deoxynucleotides (Gibco BRL, Life Technologies), 40 units of RNase inhibitor, and 400 units of SuperScript II reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). At the end of the incubation period, the enzyme was inactivated by heating at 65°C for 10 min [29]. Polymerase chain reaction PCR amplification was carried out in a 25 μl reaction mixture containing 1 × GeneAmp PCR buffer II (Perkin Elmer, Foster City, CA), 1.25 units AmpliTaq Gold DNA polymerase (Perkin Elmer), 2 mM MgCl2 (Perkin Elmer), 200 μM dNTPs (Gibco BRL, Life Technologies) and 0.5 μM of each specific primer (Table 1). The mixture was prepared before the addition of 1.25 μl of cDNA. PCR amplification was carried out using an MJResearch (Boston, MA) programmable thermal controller. The amplification program consisted of an initial denaturation step at 94°C, which was extended to 9 min in order to activate AmpliTaq Gold enzyme. The remaining cycles were 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The number of cycles performed was 27 for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 30 for CD59, C1-inh and MCP, and 32 for DAF. After amplification, PCR products were separated on a 6% polyacrylamide gel and visualized by incubation for 10 min in a solution containing 10 ng/ml of ethidium bromide. Polaroid photographs of the gels were taken. Table 1 Oligonucleotide primers used for PCR, and the corresponding restriction endonucleases used for product confirmation. Gene Sequence (5' → 3') Fragment size (introns) Genbank accession No Restriction enzymes used and the expected sizes of digestion products (bp) C1 inh-F GTT GGG GGA TGC TTT GGT AGA TTT C 332 M13690 Sau 3AI (246, 86) C1 inh-R TTA GGA CTC TGG GGC TGC TGC TGT A (2 introns) CD59-F CTG CTG CTC GTC CTG GCT GTC TTC T 280 M34671 Pst I (233, 47) CD59-R TCC CAC CAT TTT CAA GCT GTT CGT T (2 introns) MCP-F CAA TTC AGT GTG GAG TCG TGC TGC 265 Y00651 Sau 3AI (193, 72) MCP-R TGA GGC ACT GGA CGC TGG AGA T (unknown) DAF-F GTA CTG TGA ATA ATG ATG AAG GAG 364 M30142 Hae III (330, 34) DAF-R TCT TAA CTC TTC TTT GGC TAA GTC (unknown) G3PDH-F CCA TGT TCG TCA TGG GTG TGA ACC A 251 X01677 Dde I (168, 83) G3PDH-R GCC AGT AGA GGC AGG GAT GAT GTT C (2 introns) PCR primer design and restriction analyses Primers were designed to span introns so that cDNA-derived PCR products would be of different sizes to those produced if genomic DNA was amplified (see Table 1). DAF and MCP were exceptions, since only cDNA sequences were available. Primers were synthesized either by Sigma-Aldrich or ID Labs (London, ON, Canada). The primer sequences and predicted PCR fragment sizes are listed in Table 1, along with the names of the enzymes used for restriction digest analysis of each PCR fragment. The restriction digestion reactions were carried out at 37°C for 2 hr in the presence of 1 × the appropriate buffer provided by the suppliers (Invitrogen, Life Technologies and New England Biolabs, Mississauga, ON, Canada). The digested PCR products were analyzed on a 6% polyacrylamide gel (data not shown). In all cases the restriction fragments observed were of the predicted size (see Table 1). Statistical analysis The data are presented as means ± s.e.m. The significance of difference between values was estimated by means of one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test. P < 0.05 was considered to show statistically significant differences. Double fluorescence immunocytochemical analysis Oligodendrocytes, astrocytes, and microglia were harvested and air-dried on glass slides. Cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. For inactivation of endogenous peroxidase, cells were incubated with 0.3% H2O2 for 30 min. Blocking was performed for 1 hr at room temperature in 5% skim milk. For double fluorescence immunostaining, cells were incubated at room temperature overnight with a primary antibody in 1% normal serum. The primary antibody and the dilution used in the first cycle were as follows: O4 (Chemicon International, 1: 100) for oligodendrocytes, GFAP (Dako, 1: 10,000) for astrocytes, CD68 (DAKO, 1: 50) for microglia. Cells were then treated for 2 hr at room temperature with a biotin conjugated anti-mouse IgM (Vector Laboratories, Burlingame, CA, 1: 200) secondary antibody for O4, a biotin conjugated anti-rabbit IgG (Vector Laboratories, 1: 200) secondary antibody for GFAP and a biotin conjugated anti-mouse IgG (Vector Laboratories, 1: 200) secondary antibody for CD68. Then cells were incubated with Texas Red Avidin DCS (Vector Laboratories) for 1 hr. The primary antibody and the dilution used in the second cycle were as follows: for C1-inh, goat anti-C1-inhbitor (Quidel, San Diego, CA, 1: 50); for CD59, mouse anti-CD59 (Serotec Ltd, Oxford, UK, 1: 10) or rat anti-CD59 (Serotec, 1: 25). Cells were incubated at 4°C for 3 days with a primary antibody in 1% serum corresponding to the secondary antibody type. Cells were then treated for 2 hr at room temperature with FITC-conjugated anti-mouse IgG (Vector Laboratories, 1: 200), anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, 1: 200), or anti-rat IgG (Cappel, Durham, NC, 1: 200). The glass slides were then rinsed with distilled water, and a drop of Vectashield mounting medium (Vector Laboratories) placed on the slide. Results RT-PCR RT-PCR was carried out using primers for C1-inh, CD59, DAF and MCP. The housekeeping gene G3PDH was amplified in parallel with each RT-PCR run as an internal standard. Figure 1 illustrates the bands obtained for each of the RT-PCR products from oligodendrocytes (Fig. 1A), astrocytes (Fig. 1B) and microglia (Fig. 1C). Specificity of each of the products was established by endonuclease digestion (Table 1). Figure 1 Demonstration of RT-PCR products. Polaroid photographs of typical ethidium bromide-stained gels of RT-PCR products from oligodendrocytic (Fig. 1A), astrocytic (Fig. 1B) and microglial (Fig. 1C) RNA extracts. Lanes for individual mRNA products are indicated in the legend at the top. Size markers are in the right lanes. MCP, membrane cofactor protein (265 bp); DAF, decay-accelerating factor (364 bp); CD59 (280 bp); C1-inh, C1-esterase inhibitor (332 bp); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (251 bp). Semi-quantitative RT-PCR analysis To compare the ratio of each of the complement inhibitors to G3PDH, statistical analysis was performed by means of one-way ANOVA with Fisher's LSD post-hoc test (Fig. 2). The overall mean ± s.e.m. for C1-inh/G3PDH was 0.55 ± 0.12 (N = 5) in astrocytes, 0.58 ± 0.09 (N = 3) in microglia and 0.09 ± 0.06 (N = 12) in oligodendrocytes (Fig. 2A). Oligodendrocytes showed a highly significant difference from astrocytes and microglia (Fig. 2A; P < 0.001 by one-way ANOVA with Fisher's LSD post-hoc test). For MCP/G3PDH, the ratios were 0.80 ± 0.22 (N = 5) in astrocytes, 0.93 ± 0.10 (N = 3) in microglia and 0.44 ± 0.19 (N = 12) in oligodendrocytes. Oligodendrocytes showed a significant difference from astrocytes and microglia (Fig. 2B; P = 0.002 vs. astrocytes and P = 0.001 vs. microglia by one-way ANOVA with Fisher's LSD post-hoc test). The corresponding means for CD59/G3PDH were 0.73 ± 0.10 (N = 5) in astrocytes, 0.83 ± 0.03 (N = 3) in microglia and 0.76 ± 0.09 (N = 14) in oligodendrocytes (Fig. 2C). The corresponding means for DAF/G3PDH were 0.67 ± 0.07 (N = 5) in astrocytes, 0.67 ± 0.07 (N = 3) in microglia and 0.66 ± 0.15 (N = 14) in oligodendrocytes (Fig. 2D). There were no significant differences between the three cell types for CD59 and DAF. Each N represents a different patient. Figure 2 A comparison of relative complement inhibitor expression level between oligodendrocytes, astrocytes and microglia. The data were estimated by one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test (A and B; P < 0.05 was considered to show statistically significant differences). Double fluorescence immunohistochemistry In order to establish identity between oligodendroglial cells, astrocytes or microglia and cells expressing the complement inhibitor proteins CD59 or C1-inh, double fluorescence immunostaining was carried out. Oligodendrocytes were detected by O4 staining with a Texas Red tagged secondary antibody (Fig. 3A and 3D) in the first cycle and CD59 (Fig 3B) or C1-inh staining (Fig. 3E) detected with a green FITC tagged antibody in the second cycle. Astrocytes were detected by GFAP staining with a Texas Red tagged secondary antibody (Fig. 3G and 3J) in the first cycle and CD59 staining (Fig 3H) or C1-inh staining (Fig. 3K) detected with a green FITC tagged antibody in the second cycle. Microglia were detected by CD68 staining with a Texas Red tagged secondary antibody (Fig. 3M and 3P) in the first cycle, and CD59 staining (Fig 3N) or C1-inh staining (Fig. 3Q) detected with a green FITC tagged antibody in the second cycle. With double fluorescent excitation, all cells fluoresced yellow (Fig. 3C,3F,3I,3L,3O,3R), indicating colocalization of O4 with CD59 or C1-inh, GFAP with CD59 or C1-inh, and CD68 with CD59 or C1-inh. Figure 3 Double fluorescence immunohistochemistry of oligodendrocytes, astrocytes and microglia. Double fluorescence immunostaining for O4 and CD59 or C1-inh is demonstrated in A-F. In A and D, cells of typical oligodendroglial morphology were stained in the initial cycle for the specific oligodendroglial marker O4. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (B) and C1-inh (E) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In C and F, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of O4 with CD59 or C1-inh. Double fluorescence immunostaining of astrocytes for GFAP and CD59 or C1-inh is demonstrated in G-L. In G and J, cells of typical astrocytic morphology are stained in the initial cycle for the specific astroglial marker GFAP. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (H) and C1-inh (K) is shown with an FITC-linked green fluorescent secondary antibody. In I and L, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of GFAP with CD59 or C1-inh. Double fluorescence immunostaining for microglia using the specific marker CD68 and CD59 or C1-inh is demonstrated in M-R. In M and P, cells of typical microglial morphology are stained by CD68 with detection by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (N) and C1-inh (Q) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In O and R, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of CD68 with CD59 or C1-inh. (Magnification: × 200) Discussion This report shows that human oligodendrocytes express a much lower level of mRNA for C1-inh than astrocytes and microglia, and a significantly lower level of mRNA for MCP. The mRNA levels of CD59 and DAF were comparable in all the three cell types. Overall our data suggest that oligodendroglial cells, in common with other cell types, can produce complement inhibitors, but at a significantly lower level for C1-inh and MCP. It has already been reported that human neurons and Schwann cells [24], neuroblastoma cell lines [18], astrocytes [23], astrocytoma cell lines [25,26], the HOG human oligodendroglial cell line [1] and oligodendrocytes [21,22] produce some or all of the complement inhibitor proteins and their mRNAs. Activation of the complement cascade and deposition of activated complement fragments occur in non-infectious diseases such as multiple sclerosis, Pick's disease, Alzheimer's disease and other neurodegenerative conditions [15,16,30-34]. Complement inhibitors may play an important role in preventing such pathology. Full activation of the complement cascade requires overcoming a series of endogenous inhibitory factors. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis [35-37] and this vulnerability may be related to a deficiency of their ability to express complement regulatory proteins [22], particularly C1-inh. Sporadic complement attack, in the form of complement activated oligodendroglia (CAO) is also observed in a number of neurodegenerative conditions [38,39], including Alzheimer's, Pick's, Huntington's and Parkinson's diseases, amyotrophic lateral sclerosis, progressive supranuclear palsy, Shy-Drager syndrome, argyrophilic grain dementia and pallido-nigral luysial atrophy [38,39]. The source of the complement proteins that become activated is unknown, but the data presented here suggest that oligodendrocytes are vulnerable to complement attack because of a low expression of C1-inh and MCP. Conclusions These results suggest that the lower expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system, particularly multiple sclerosis. List of abbreviations analysis of variance (ANOVA) central nervous system (CNS) complement activated oligodendroglia (CAO) complement receptor 1 (CR1) decay-accelerating factor (DAF) dithiothreitol (DTT) fluorescein isothiocyanate isomer (FITC) glyceraldehyde-3-phosphate dehydrogenase (G3PDH) glial fibrillary acidic protein (GFAP) homologous restriction factor (HRF) membrane cofactor protein (MCP) phosphate-buffered saline (PBS) Competing interests None declared. Authors' contributions MH was responsible for the majority of the experimental studies, and for writing the manuscript. AK contributed to the cell culture and the editing of the manuscript. PLM contributed to the conception, interpretation of results and the writing and editing of the manuscript. Acknowledgements This work was supported by a grant from the Jack Brown and Family Alzheimer's Disease Research Fund, and the Pacific Parkinson's Research Institute. ==== Refs Gasque P Morgan BP Complement regulatory protein expression by human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes Immunology 1996 89 338 347 8958045 10.1046/j.1365-2567.1996.d01-756.x Hosokawa M Klegeris A Maguire J McGeer PL Expression of complement mRNAs and proteins by human oligodendroglial cells Glia 2003 42 417 423 12730962 10.1002/glia.10234 Levi-Strauss M Mallat M Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation J Immunol 1987 139 2361 2366 3655365 Gordon DL Avery VM Adrian DL Sadlon TA Detection of complement protein mRNA in human astrocytes by polymerase chain reaction J Neurosci Methods 1992 45 191 197 1294852 10.1016/0165-0270(92)90076-P Johnson SA Lampert-Etchells M Pasinetti GM Rozovsky I Finch CE Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning Neurobiol Aging 1992 13 641 648 1362796 10.1016/0197-4580(92)90086-D Pasinetti GM Johnson SA Rozovsky I Lampert-Etchells M Morgan DG Gordon MN Morgan TE Willoughby D Finch CE Complement C1qB and C4 mRNAs responses to lesioning in rat brain Exp Neurol 1992 118 117 125 1426121 10.1016/0014-4886(92)90028-O Rus HG Kim LM Niculescu FI Shin ML Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus J Immunol 1992 148 928 933 1530957 Haga S Ikeda K Sato M Ishii T Synthetic Alzheimer amyloid β/A4 peptides enhance production of complement C3 component by cultured microglial cells Brain Res 1993 601 88 94 8431789 10.1016/0006-8993(93)91698-R Gasque P Julen N Ischenko AM Picot C Mauger C Chauzy C Ripoche J Fontaine M Expression of complement components of the alternative pathway by glioma cell lines J Immunol 1992 149 1381 1387 1386864 Gasque P Ischenko A Legoedec J Mauger C Schouft MT Fontaine M Expression of the complement classical pathway by human glioma in culture J Biol Chem 1993 268 25068 25074 8227070 Gasque P Fontaine M Morgan BP Complement expression in human brain J Immunol 1995 154 4726 4733 7536777 Barnum SR Complement biosynthesis in the central nervous system Crit Rev Oral Biol Med 1995 6 132 146 7548620 Walker DG Kim SU McGeer PL Complement and cytokine gene expression in cultured microglia derived from postmortem human brains J Neurosci Res 1995 40 478 493 7616608 Walker DG Kim SU McGeer PL Expression of complement C4 and C9 genes by human astrocytes Brain Res 1998 809 31 38 9795119 10.1016/S0006-8993(98)00811-7 Terai K Walker DG McGeer EG McGeer PL Neurons express proteins of the classical complement pathway in Alzheimer disease Brain Res 1997 769 385 390 9374211 10.1016/S0006-8993(97)00849-4 Shen Y Li R McGeer EG McGeer PL Neuronal expression of mRNAs for complement proteins of the classical pathway in Alzheimer brain Brain Res 1997 769 391 395 9374212 10.1016/S0006-8993(97)00850-0 Gasque P Thomas A Fontaine M Morgan BP Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins J Neuroimmunol 1996 66 29 40 8964911 10.1016/0165-5728(96)00015-X Thomas A Gasque P Vaudry D Gonzalez B Fontaine M Expression of a complete and functional complement system by human neuronal cells in vitro Int Immunol 2000 12 1015 1023 10882413 10.1093/intimm/12.7.1015 Vastag M Skopal J Kramer J Kolev K Voko Z Csonka E Machovich R Nagy Z Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4 Immunobiology 1998 199 5 13 9717663 Klegeris A Bissonnette CJ Dorovini-Zis K McGeer PL Expression of complement messenger RNAs by human endothelial cells Brain Res 2000 871 1 6 10882776 10.1016/S0006-8993(00)02253-8 Zajicek J Wing M Skepper J Compston A Human oligodendrocytes are not sensitive to complement. A study of CD59 expression in the human central nervous system Lab Invest 1995 73 128 138 7541492 Scolding NJ Morgan BP Compston DAS The expression of complement regulatory proteins by adult human oligodendrocytes J Neuroimmunol 1998 84 69 75 9600710 10.1016/S0165-5728(97)00241-5 Gordon DL Sadlon T Hefford C Adrian D Expression of CD59, a regulator of the membrane attack complex of complement, on human astrocytes Brain Res Mol Brain Res 1993 18 335 338 7686996 10.1016/0169-328X(93)90098-A Vedeler C Ulvestad E Bjorge L Conti G Williams K Mork S Matre R The expression of CD59 in normal human nervous tissue Immunology 1994 82 542 547 7530684 Yang C Jones JL Barnum SR Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes J Neuroimmunol 1993 47 123 132 7690370 10.1016/0165-5728(93)90022-Q Spiller OB Moretto G Kim SU Morgan BP Devine DV Complement expression on astrocytes and astrocytoma cell lines failure of complement regulation at the C3 level correlates with very low CD59 expression J Neuroimmunol 1996 71 97 106 8982108 10.1016/S0165-5728(96)00136-1 De Groot CJA Langeveld CH Jongenelen CAM Montagne L Van Der Valk P Dijkstra C Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization J Neurosci Res 1997 49 342 254 9260745 10.1002/(SICI)1097-4547(19970801)49:3<342::AID-JNR9>3.0.CO;2-C De Groot CJA Montagne L Janssen I Ravid R Van Der Valk P Veerhuis R Isolation and characterization of adult microglial cells and oligodendrocytes derived from postmortem human brain tissue Brain Res Brain Res Protoc 2000 5 85 94 10719269 10.1016/S1385-299X(99)00059-8 Yasojima K Schwab C McGeer EG McGeer PL Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease Brain Res 2000 887 80 89 11134592 10.1016/S0006-8993(00)02970-X Eikelenboom P Hack CE Rozemuller JM Stam FC Complement activation in amyloid plaques in Alzheimer's dementia Virchows Arch B Cell Pathol Incl Mol Pathol 1989 56 259 262 2565620 McGeer PL Akiyama H Itagaki S McGeer EG Immune system response in Alzheimer's disease Can J Neurol Sci 1989 16 516 527 2804814 Verga L Frangione B Tagliavini F Giaccone G Migheli A Bugiani O Alzheimer patients and Down patients: cerebral preamyloid deposits differ ultrastructurally and histochemically from the amyloid of senile plaques Neurosci Lett 1989 105 294 299 2531851 10.1016/0304-3940(89)90636-8 McGeer PL McGeer EG The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases Brain Res Rev 1995 21 195 218 8866675 10.1016/0165-0173(95)00011-9 Webster S Lue LF Brachova L Tenner AJ McGeer PL Terai K Walker DG Bradt B Cooper NR Rogers J Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease Neurobiol Aging 1997 18 415 421 9330973 10.1016/S0197-4580(97)00042-0 Compston DAS Morgan BP Campbell AK Wilkins P Cole G Thomas ND Jasani B Immunocytochemical localization of the terminal complement complex in multiple sclerosis Neuropathol Appl Neurobiol 1989 15 307 316 2779734 Prineas JW Kwon EE Cho ES Sharer LR Barnett MH Oleszak EL Hoffman B Morgan BP Immunopathology of secondary-progressive multiple sclerosis Ann Neurol 2001 50 646 657 11706971 10.1002/ana.1255 Schwab C McGeer PL Complement activated C4d immunoreactive oligodendrocytes delineate small cortical plaques in multiple sclerosis Exp Neurol 2002 174 81 88 11869036 10.1006/exnr.2001.7851 Yamada T Akiyama H McGeer PL Complement-activated oligodendroglia: a new pathogenic entity identified by immunostaining with antibodies to human complement proteins C3d and C4d Neurosci Lett 1990 112 161 166 2359515 10.1016/0304-3940(90)90196-G Yamada T McGeer PL McGeer EG Relationship of complement-activated oligodendrocytes to reactive microglia and neuronal pathology in neurodegenerative disease Dementia 1991 2 71 77

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J Neuroinflammation

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==== Front J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central London 1742-2094-1-171532769010.1186/1742-2094-1-17ResearchHuman oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAs Hosokawa Masato [email protected] Andis [email protected] Patrick L [email protected] Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, V6T 1Z3, Canada2004 24 8 2004 1 17 17 20 5 2004 24 8 2004 Copyright © 2004 Hosokawa et al; licensee BioMed Central Ltd.2004Hosokawa et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis. This vulnerability may be related to a deficiency in their ability to express complement regulatory proteins. Methods This study compared the expression level of complement inhibitor mRNAs by human oligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR. Results Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expression was dramatically lower in oligodendroglial cells compared with astrocytes and microglia. The mRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was also significantly lower than for other cell types. Conclusion The lower mRNA expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system. ==== Body Background Resident brain cells including oligodendrocytes [1,2], astrocytes, astrocytomas, microglia, glioblastomas [3-14], neurons [15,16], neuroblastomas [17,18] and endothelial cells [19,20] express mRNAs for complement proteins. Although the role of complement expression by these cells remains unclear, local complement activation in the central nervous system (CNS) might damage these cells and contribute to the pathology in several inflammatory and neurodegenerative diseases including multiple sclerosis, Alzheimer's disease and progressive supranuclear palsy. For self-protection, resident brain cells also express complement inhibitors, such as membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligodendroglial cell line produces MCP, DAF, CD59, C1-inh and S-protein, but not complement receptor 1 (CR1) [1]. Human oligodendrocytes have been reported to express CD59 [21] and DAF, but not MCP, CR1, homologous restriction factor (HRF: C8 bp) or clusterin [22]. Astrocytes [23], neurons and Schwann cells have been reported to express CD59 [24] and neuroblastoma cell lines C1-inh [18]. Astrocytoma cell lines have been reported to express MCP, DAF, and CD59 [25,26]. In this study, the expression level of mRNAs for various complement inhibitors by human oligodendrocytes, astrocytes and microglia were compared by semi-quantitative PCR. We show that oligodendrocytes express extremely low levels of mRNA for C1-inh and significantly lower levels of mRNA for MCP compared with astrocytes and microglia. The expression level of mRNAs for CD59 and DAF showed no significant differences between the three cell types. Methods Cell culture: microglial- and astrocyte-enriched cultures Human microglial and astrocytic cells were isolated from surgically resected temporal lobe tissues. We thank Dr. J. Maguire, Department of Pathology and Laboratory Medicine, Vancouver General Hospital for providing the surgical specimens. Isolation protocols described by De Groot et al. [27,28] were used with minor modifications. Tissues were placed in a sterile Petri dish, rinsed with Hank's balanced salt solution, and visible blood vessels were removed. After washing tissues two more times with Hank's balanced salt solution, tissues were chopped into small (<2 mm3) pieces with a sterile scalpel. The fragments were transferred into a 50 ml centrifuge tube containing 10 ml of 0.25% trypsin solution (Gibco-BRL, Life Technologies, Burlington, ON, Canada), and incubated at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d'Urfé, PQ, Canada) was added to reach a final concentration of 50 μg/ml. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F12 ham (DMEM-F12; Sigma-Aldrich, Oakville, ON, Canada) with 10% fetal bovine serum (FBS; Gibco-BRL, Life Technologies), and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 × g for 10 min, the cell pellet was resuspended in serum containing medium, triturated several times, and passed through a 100 μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged once more (275 × g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin (50 μg/ml, from Sigma), and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO2, 95% air atmosphere at 37°C for 2 hr in order to achieve adherence of microglial cells. Non-adherent cells with myelin debris were removed from these microglia-enriched cultures and transferred into poly-L-lysine coated 10 cm tissue culture plates in order to achieve adherence of astrocytes. Plates were incubated for 48 hr, after which the culture medium containing myelin debris and non-adherent cells was removed and used to prepare oligodendroglial cell cultures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before their mRNAs were extracted. Immunostaining with antibodies against CD68 (Dako, Mississauga, ON, Canada) which stains microglia as well as macrophages, and glial fibrillary acidic protein (GFAP, Dako), which is a marker of astrocytes, showed that the microglia-enriched cultures contained 93.5 ± 3.6 % (N = 4) microglial cells, while astrocyte-enriched cultures contained 85.7 ± 3.4 % (N = 4) astrocytes. Cell culture: oligodendroglial cells These were prepared as described before [2]. Briefly, cell culture media containing myelin debris and non-adherent cells that were removed from astrocyte-enriched cultures were used to extract oligodendroglial cells. The non-adherent cells were collected by centrifugation at 275 × g for 10 min and replated onto uncoated 10 cm tissue culture plates for another 24 hr. Subsequently, the cell culture medium containing floating cells was transferred to 50 ml tubes and Lymphoprep solution (Axis-Shield, Oslo, Norway) used to reduce the amount of contaminating myelin debris. For this purpose, 10 ml of Lymphoprep solution was carefully placed under the oligodendrocyte cell suspension and the density gradient was centrifuged at 325 × g for 10 min. The interphase was collected and transferred to a 50 ml centrifuge tube. Fresh culture medium was added and the suspension was centrifuged at 275 × g for 7 min. The cell pellet was resuspended and the oligodendrocyte cultures seeded onto 60 mm plastic culture dishes. Immunostaining with anti-O4 antibody (Chemicon International, Temecula, CA), which is a marker of oligodendrocytes, showed that the oligodendrocytes-enriched cultures contained 95.3 ± 4.4 % (N = 4) oligodendrocytes. RNA isolation and cDNA synthesis by reverse transcription Total RNA from oligodendroglial cells, microglia, and astrocytes were isolated by the acid guanidium thiocyanate-phenol-chloroform method. Two μg of the RNA was then used to prepare cDNA. RNA was treated with 10 U of DNase I (Gibco BRL, Life Technologies) for 60 min at 37°C in 25 μl of 1 × reverse transcriptase buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2) containing 40 U of RNase inhibitor (Pharmacia Biotech) and 1 mM dithiothreitol (DTT), following by incubation at 85°C for 5 min to inactivate the enzyme. Reverse transcription was performed at 42°C for 90 min in 50 μl of the following mixture: 1 × reverse transcriptase buffer containing 2 μg of RNA, 5 mM DTT, 0.2 μg random hexamer primers (Pharmacia Biotech), 1 mM deoxynucleotides (Gibco BRL, Life Technologies), 40 units of RNase inhibitor, and 400 units of SuperScript II reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). At the end of the incubation period, the enzyme was inactivated by heating at 65°C for 10 min [29]. Polymerase chain reaction PCR amplification was carried out in a 25 μl reaction mixture containing 1 × GeneAmp PCR buffer II (Perkin Elmer, Foster City, CA), 1.25 units AmpliTaq Gold DNA polymerase (Perkin Elmer), 2 mM MgCl2 (Perkin Elmer), 200 μM dNTPs (Gibco BRL, Life Technologies) and 0.5 μM of each specific primer (Table 1). The mixture was prepared before the addition of 1.25 μl of cDNA. PCR amplification was carried out using an MJResearch (Boston, MA) programmable thermal controller. The amplification program consisted of an initial denaturation step at 94°C, which was extended to 9 min in order to activate AmpliTaq Gold enzyme. The remaining cycles were 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The number of cycles performed was 27 for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 30 for CD59, C1-inh and MCP, and 32 for DAF. After amplification, PCR products were separated on a 6% polyacrylamide gel and visualized by incubation for 10 min in a solution containing 10 ng/ml of ethidium bromide. Polaroid photographs of the gels were taken. Table 1 Oligonucleotide primers used for PCR, and the corresponding restriction endonucleases used for product confirmation. Gene Sequence (5' → 3') Fragment size (introns) Genbank accession No Restriction enzymes used and the expected sizes of digestion products (bp) C1 inh-F GTT GGG GGA TGC TTT GGT AGA TTT C 332 M13690 Sau 3AI (246, 86) C1 inh-R TTA GGA CTC TGG GGC TGC TGC TGT A (2 introns) CD59-F CTG CTG CTC GTC CTG GCT GTC TTC T 280 M34671 Pst I (233, 47) CD59-R TCC CAC CAT TTT CAA GCT GTT CGT T (2 introns) MCP-F CAA TTC AGT GTG GAG TCG TGC TGC 265 Y00651 Sau 3AI (193, 72) MCP-R TGA GGC ACT GGA CGC TGG AGA T (unknown) DAF-F GTA CTG TGA ATA ATG ATG AAG GAG 364 M30142 Hae III (330, 34) DAF-R TCT TAA CTC TTC TTT GGC TAA GTC (unknown) G3PDH-F CCA TGT TCG TCA TGG GTG TGA ACC A 251 X01677 Dde I (168, 83) G3PDH-R GCC AGT AGA GGC AGG GAT GAT GTT C (2 introns) PCR primer design and restriction analyses Primers were designed to span introns so that cDNA-derived PCR products would be of different sizes to those produced if genomic DNA was amplified (see Table 1). DAF and MCP were exceptions, since only cDNA sequences were available. Primers were synthesized either by Sigma-Aldrich or ID Labs (London, ON, Canada). The primer sequences and predicted PCR fragment sizes are listed in Table 1, along with the names of the enzymes used for restriction digest analysis of each PCR fragment. The restriction digestion reactions were carried out at 37°C for 2 hr in the presence of 1 × the appropriate buffer provided by the suppliers (Invitrogen, Life Technologies and New England Biolabs, Mississauga, ON, Canada). The digested PCR products were analyzed on a 6% polyacrylamide gel (data not shown). In all cases the restriction fragments observed were of the predicted size (see Table 1). Statistical analysis The data are presented as means ± s.e.m. The significance of difference between values was estimated by means of one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test. P < 0.05 was considered to show statistically significant differences. Double fluorescence immunocytochemical analysis Oligodendrocytes, astrocytes, and microglia were harvested and air-dried on glass slides. Cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. For inactivation of endogenous peroxidase, cells were incubated with 0.3% H2O2 for 30 min. Blocking was performed for 1 hr at room temperature in 5% skim milk. For double fluorescence immunostaining, cells were incubated at room temperature overnight with a primary antibody in 1% normal serum. The primary antibody and the dilution used in the first cycle were as follows: O4 (Chemicon International, 1: 100) for oligodendrocytes, GFAP (Dako, 1: 10,000) for astrocytes, CD68 (DAKO, 1: 50) for microglia. Cells were then treated for 2 hr at room temperature with a biotin conjugated anti-mouse IgM (Vector Laboratories, Burlingame, CA, 1: 200) secondary antibody for O4, a biotin conjugated anti-rabbit IgG (Vector Laboratories, 1: 200) secondary antibody for GFAP and a biotin conjugated anti-mouse IgG (Vector Laboratories, 1: 200) secondary antibody for CD68. Then cells were incubated with Texas Red Avidin DCS (Vector Laboratories) for 1 hr. The primary antibody and the dilution used in the second cycle were as follows: for C1-inh, goat anti-C1-inhbitor (Quidel, San Diego, CA, 1: 50); for CD59, mouse anti-CD59 (Serotec Ltd, Oxford, UK, 1: 10) or rat anti-CD59 (Serotec, 1: 25). Cells were incubated at 4°C for 3 days with a primary antibody in 1% serum corresponding to the secondary antibody type. Cells were then treated for 2 hr at room temperature with FITC-conjugated anti-mouse IgG (Vector Laboratories, 1: 200), anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, 1: 200), or anti-rat IgG (Cappel, Durham, NC, 1: 200). The glass slides were then rinsed with distilled water, and a drop of Vectashield mounting medium (Vector Laboratories) placed on the slide. Results RT-PCR RT-PCR was carried out using primers for C1-inh, CD59, DAF and MCP. The housekeeping gene G3PDH was amplified in parallel with each RT-PCR run as an internal standard. Figure 1 illustrates the bands obtained for each of the RT-PCR products from oligodendrocytes (Fig. 1A), astrocytes (Fig. 1B) and microglia (Fig. 1C). Specificity of each of the products was established by endonuclease digestion (Table 1). Figure 1 Demonstration of RT-PCR products. Polaroid photographs of typical ethidium bromide-stained gels of RT-PCR products from oligodendrocytic (Fig. 1A), astrocytic (Fig. 1B) and microglial (Fig. 1C) RNA extracts. Lanes for individual mRNA products are indicated in the legend at the top. Size markers are in the right lanes. MCP, membrane cofactor protein (265 bp); DAF, decay-accelerating factor (364 bp); CD59 (280 bp); C1-inh, C1-esterase inhibitor (332 bp); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (251 bp). Semi-quantitative RT-PCR analysis To compare the ratio of each of the complement inhibitors to G3PDH, statistical analysis was performed by means of one-way ANOVA with Fisher's LSD post-hoc test (Fig. 2). The overall mean ± s.e.m. for C1-inh/G3PDH was 0.55 ± 0.12 (N = 5) in astrocytes, 0.58 ± 0.09 (N = 3) in microglia and 0.09 ± 0.06 (N = 12) in oligodendrocytes (Fig. 2A). Oligodendrocytes showed a highly significant difference from astrocytes and microglia (Fig. 2A; P < 0.001 by one-way ANOVA with Fisher's LSD post-hoc test). For MCP/G3PDH, the ratios were 0.80 ± 0.22 (N = 5) in astrocytes, 0.93 ± 0.10 (N = 3) in microglia and 0.44 ± 0.19 (N = 12) in oligodendrocytes. Oligodendrocytes showed a significant difference from astrocytes and microglia (Fig. 2B; P = 0.002 vs. astrocytes and P = 0.001 vs. microglia by one-way ANOVA with Fisher's LSD post-hoc test). The corresponding means for CD59/G3PDH were 0.73 ± 0.10 (N = 5) in astrocytes, 0.83 ± 0.03 (N = 3) in microglia and 0.76 ± 0.09 (N = 14) in oligodendrocytes (Fig. 2C). The corresponding means for DAF/G3PDH were 0.67 ± 0.07 (N = 5) in astrocytes, 0.67 ± 0.07 (N = 3) in microglia and 0.66 ± 0.15 (N = 14) in oligodendrocytes (Fig. 2D). There were no significant differences between the three cell types for CD59 and DAF. Each N represents a different patient. Figure 2 A comparison of relative complement inhibitor expression level between oligodendrocytes, astrocytes and microglia. The data were estimated by one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test (A and B; P < 0.05 was considered to show statistically significant differences). Double fluorescence immunohistochemistry In order to establish identity between oligodendroglial cells, astrocytes or microglia and cells expressing the complement inhibitor proteins CD59 or C1-inh, double fluorescence immunostaining was carried out. Oligodendrocytes were detected by O4 staining with a Texas Red tagged secondary antibody (Fig. 3A and 3D) in the first cycle and CD59 (Fig 3B) or C1-inh staining (Fig. 3E) detected with a green FITC tagged antibody in the second cycle. Astrocytes were detected by GFAP staining with a Texas Red tagged secondary antibody (Fig. 3G and 3J) in the first cycle and CD59 staining (Fig 3H) or C1-inh staining (Fig. 3K) detected with a green FITC tagged antibody in the second cycle. Microglia were detected by CD68 staining with a Texas Red tagged secondary antibody (Fig. 3M and 3P) in the first cycle, and CD59 staining (Fig 3N) or C1-inh staining (Fig. 3Q) detected with a green FITC tagged antibody in the second cycle. With double fluorescent excitation, all cells fluoresced yellow (Fig. 3C,3F,3I,3L,3O,3R), indicating colocalization of O4 with CD59 or C1-inh, GFAP with CD59 or C1-inh, and CD68 with CD59 or C1-inh. Figure 3 Double fluorescence immunohistochemistry of oligodendrocytes, astrocytes and microglia. Double fluorescence immunostaining for O4 and CD59 or C1-inh is demonstrated in A-F. In A and D, cells of typical oligodendroglial morphology were stained in the initial cycle for the specific oligodendroglial marker O4. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (B) and C1-inh (E) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In C and F, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of O4 with CD59 or C1-inh. Double fluorescence immunostaining of astrocytes for GFAP and CD59 or C1-inh is demonstrated in G-L. In G and J, cells of typical astrocytic morphology are stained in the initial cycle for the specific astroglial marker GFAP. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (H) and C1-inh (K) is shown with an FITC-linked green fluorescent secondary antibody. In I and L, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of GFAP with CD59 or C1-inh. Double fluorescence immunostaining for microglia using the specific marker CD68 and CD59 or C1-inh is demonstrated in M-R. In M and P, cells of typical microglial morphology are stained by CD68 with detection by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (N) and C1-inh (Q) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In O and R, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of CD68 with CD59 or C1-inh. (Magnification: × 200) Discussion This report shows that human oligodendrocytes express a much lower level of mRNA for C1-inh than astrocytes and microglia, and a significantly lower level of mRNA for MCP. The mRNA levels of CD59 and DAF were comparable in all the three cell types. Overall our data suggest that oligodendroglial cells, in common with other cell types, can produce complement inhibitors, but at a significantly lower level for C1-inh and MCP. It has already been reported that human neurons and Schwann cells [24], neuroblastoma cell lines [18], astrocytes [23], astrocytoma cell lines [25,26], the HOG human oligodendroglial cell line [1] and oligodendrocytes [21,22] produce some or all of the complement inhibitor proteins and their mRNAs. Activation of the complement cascade and deposition of activated complement fragments occur in non-infectious diseases such as multiple sclerosis, Pick's disease, Alzheimer's disease and other neurodegenerative conditions [15,16,30-34]. Complement inhibitors may play an important role in preventing such pathology. Full activation of the complement cascade requires overcoming a series of endogenous inhibitory factors. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis [35-37] and this vulnerability may be related to a deficiency of their ability to express complement regulatory proteins [22], particularly C1-inh. Sporadic complement attack, in the form of complement activated oligodendroglia (CAO) is also observed in a number of neurodegenerative conditions [38,39], including Alzheimer's, Pick's, Huntington's and Parkinson's diseases, amyotrophic lateral sclerosis, progressive supranuclear palsy, Shy-Drager syndrome, argyrophilic grain dementia and pallido-nigral luysial atrophy [38,39]. The source of the complement proteins that become activated is unknown, but the data presented here suggest that oligodendrocytes are vulnerable to complement attack because of a low expression of C1-inh and MCP. Conclusions These results suggest that the lower expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system, particularly multiple sclerosis. List of abbreviations analysis of variance (ANOVA) central nervous system (CNS) complement activated oligodendroglia (CAO) complement receptor 1 (CR1) decay-accelerating factor (DAF) dithiothreitol (DTT) fluorescein isothiocyanate isomer (FITC) glyceraldehyde-3-phosphate dehydrogenase (G3PDH) glial fibrillary acidic protein (GFAP) homologous restriction factor (HRF) membrane cofactor protein (MCP) phosphate-buffered saline (PBS) Competing interests None declared. Authors' contributions MH was responsible for the majority of the experimental studies, and for writing the manuscript. AK contributed to the cell culture and the editing of the manuscript. PLM contributed to the conception, interpretation of results and the writing and editing of the manuscript. Acknowledgements This work was supported by a grant from the Jack Brown and Family Alzheimer's Disease Research Fund, and the Pacific Parkinson's Research Institute. ==== Refs Gasque P Morgan BP Complement regulatory protein expression by human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes Immunology 1996 89 338 347 8958045 10.1046/j.1365-2567.1996.d01-756.x Hosokawa M Klegeris A Maguire J McGeer PL Expression of complement mRNAs and proteins by human oligodendroglial cells Glia 2003 42 417 423 12730962 10.1002/glia.10234 Levi-Strauss M Mallat M Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation J Immunol 1987 139 2361 2366 3655365 Gordon DL Avery VM Adrian DL Sadlon TA Detection of complement protein mRNA in human astrocytes by polymerase chain reaction J Neurosci Methods 1992 45 191 197 1294852 10.1016/0165-0270(92)90076-P Johnson SA Lampert-Etchells M Pasinetti GM Rozovsky I Finch CE Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning Neurobiol Aging 1992 13 641 648 1362796 10.1016/0197-4580(92)90086-D Pasinetti GM Johnson SA Rozovsky I Lampert-Etchells M Morgan DG Gordon MN Morgan TE Willoughby D Finch CE Complement C1qB and C4 mRNAs responses to lesioning in rat brain Exp Neurol 1992 118 117 125 1426121 10.1016/0014-4886(92)90028-O Rus HG Kim LM Niculescu FI Shin ML Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus J Immunol 1992 148 928 933 1530957 Haga S Ikeda K Sato M Ishii T Synthetic Alzheimer amyloid β/A4 peptides enhance production of complement C3 component by cultured microglial cells Brain Res 1993 601 88 94 8431789 10.1016/0006-8993(93)91698-R Gasque P Julen N Ischenko AM Picot C Mauger C Chauzy C Ripoche J Fontaine M Expression of complement components of the alternative pathway by glioma cell lines J Immunol 1992 149 1381 1387 1386864 Gasque P Ischenko A Legoedec J Mauger C Schouft MT Fontaine M Expression of the complement classical pathway by human glioma in culture J Biol Chem 1993 268 25068 25074 8227070 Gasque P Fontaine M Morgan BP Complement expression in human brain J Immunol 1995 154 4726 4733 7536777 Barnum SR Complement biosynthesis in the central nervous system Crit Rev Oral Biol Med 1995 6 132 146 7548620 Walker DG Kim SU McGeer PL Complement and cytokine gene expression in cultured microglia derived from postmortem human brains J Neurosci Res 1995 40 478 493 7616608 Walker DG Kim SU McGeer PL Expression of complement C4 and C9 genes by human astrocytes Brain Res 1998 809 31 38 9795119 10.1016/S0006-8993(98)00811-7 Terai K Walker DG McGeer EG McGeer PL Neurons express proteins of the classical complement pathway in Alzheimer disease Brain Res 1997 769 385 390 9374211 10.1016/S0006-8993(97)00849-4 Shen Y Li R McGeer EG McGeer PL Neuronal expression of mRNAs for complement proteins of the classical pathway in Alzheimer brain Brain Res 1997 769 391 395 9374212 10.1016/S0006-8993(97)00850-0 Gasque P Thomas A Fontaine M Morgan BP Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins J Neuroimmunol 1996 66 29 40 8964911 10.1016/0165-5728(96)00015-X Thomas A Gasque P Vaudry D Gonzalez B Fontaine M Expression of a complete and functional complement system by human neuronal cells in vitro Int Immunol 2000 12 1015 1023 10882413 10.1093/intimm/12.7.1015 Vastag M Skopal J Kramer J Kolev K Voko Z Csonka E Machovich R Nagy Z Endothelial cells cultured from human brain microvessels produce complement proteins factor H, factor B, C1 inhibitor, and C4 Immunobiology 1998 199 5 13 9717663 Klegeris A Bissonnette CJ Dorovini-Zis K McGeer PL Expression of complement messenger RNAs by human endothelial cells Brain Res 2000 871 1 6 10882776 10.1016/S0006-8993(00)02253-8 Zajicek J Wing M Skepper J Compston A Human oligodendrocytes are not sensitive to complement. A study of CD59 expression in the human central nervous system Lab Invest 1995 73 128 138 7541492 Scolding NJ Morgan BP Compston DAS The expression of complement regulatory proteins by adult human oligodendrocytes J Neuroimmunol 1998 84 69 75 9600710 10.1016/S0165-5728(97)00241-5 Gordon DL Sadlon T Hefford C Adrian D Expression of CD59, a regulator of the membrane attack complex of complement, on human astrocytes Brain Res Mol Brain Res 1993 18 335 338 7686996 10.1016/0169-328X(93)90098-A Vedeler C Ulvestad E Bjorge L Conti G Williams K Mork S Matre R The expression of CD59 in normal human nervous tissue Immunology 1994 82 542 547 7530684 Yang C Jones JL Barnum SR Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes J Neuroimmunol 1993 47 123 132 7690370 10.1016/0165-5728(93)90022-Q Spiller OB Moretto G Kim SU Morgan BP Devine DV Complement expression on astrocytes and astrocytoma cell lines failure of complement regulation at the C3 level correlates with very low CD59 expression J Neuroimmunol 1996 71 97 106 8982108 10.1016/S0165-5728(96)00136-1 De Groot CJA Langeveld CH Jongenelen CAM Montagne L Van Der Valk P Dijkstra C Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization J Neurosci Res 1997 49 342 254 9260745 10.1002/(SICI)1097-4547(19970801)49:3<342::AID-JNR9>3.0.CO;2-C De Groot CJA Montagne L Janssen I Ravid R Van Der Valk P Veerhuis R Isolation and characterization of adult microglial cells and oligodendrocytes derived from postmortem human brain tissue Brain Res Brain Res Protoc 2000 5 85 94 10719269 10.1016/S1385-299X(99)00059-8 Yasojima K Schwab C McGeer EG McGeer PL Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease Brain Res 2000 887 80 89 11134592 10.1016/S0006-8993(00)02970-X Eikelenboom P Hack CE Rozemuller JM Stam FC Complement activation in amyloid plaques in Alzheimer's dementia Virchows Arch B Cell Pathol Incl Mol Pathol 1989 56 259 262 2565620 McGeer PL Akiyama H Itagaki S McGeer EG Immune system response in Alzheimer's disease Can J Neurol Sci 1989 16 516 527 2804814 Verga L Frangione B Tagliavini F Giaccone G Migheli A Bugiani O Alzheimer patients and Down patients: cerebral preamyloid deposits differ ultrastructurally and histochemically from the amyloid of senile plaques Neurosci Lett 1989 105 294 299 2531851 10.1016/0304-3940(89)90636-8 McGeer PL McGeer EG The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases Brain Res Rev 1995 21 195 218 8866675 10.1016/0165-0173(95)00011-9 Webster S Lue LF Brachova L Tenner AJ McGeer PL Terai K Walker DG Bradt B Cooper NR Rogers J Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer's disease Neurobiol Aging 1997 18 415 421 9330973 10.1016/S0197-4580(97)00042-0 Compston DAS Morgan BP Campbell AK Wilkins P Cole G Thomas ND Jasani B Immunocytochemical localization of the terminal complement complex in multiple sclerosis Neuropathol Appl Neurobiol 1989 15 307 316 2779734 Prineas JW Kwon EE Cho ES Sharer LR Barnett MH Oleszak EL Hoffman B Morgan BP Immunopathology of secondary-progressive multiple sclerosis Ann Neurol 2001 50 646 657 11706971 10.1002/ana.1255 Schwab C McGeer PL Complement activated C4d immunoreactive oligodendrocytes delineate small cortical plaques in multiple sclerosis Exp Neurol 2002 174 81 88 11869036 10.1006/exnr.2001.7851 Yamada T Akiyama H McGeer PL Complement-activated oligodendroglia: a new pathogenic entity identified by immunostaining with antibodies to human complement proteins C3d and C4d Neurosci Lett 1990 112 161 166 2359515 10.1016/0304-3940(90)90196-G Yamada T McGeer PL McGeer EG Relationship of complement-activated oligodendrocytes to reactive microglia and neuronal pathology in neurodegenerative disease Dementia 1991 2 71 77

15347421

PMC516792

CC BY

2021-01-04 16:36:37

no

Retrovirology. 2004 Sep 3; 1:24

latin-1

Retrovirology

10.1186/1742-4690-1-24

oa_comm

==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020263Book Reviews/Science in the MediaGenetics/Genomics/Gene TherapyScience PolicyNoneParadoxes of Difference Book Reviews/Science in the MediaLee Sandra Soo-Jin 9 2004 14 9 2004 14 9 2004 2 9 e263Medley C (2004) Relativity [stage production]. San Francisco: Magic Theater. Produced Spring 2004  Copyright: © 2004 Sandra Soo-Jin Lee.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.A new play tackles the politics around race and gender in science ==== Body Can “race” be gleaned from our genes? Concern over the emerging trajectories of genetics research has led to ongoing debates about how to characterize and interpret genetic variation. Despite the mantra that humans share 99.9% of our genetic makeup, there is increasing interest in identifying the relatively small percentage difference that distinguishes individuals. Therein lies the paradox: if we are all the same, why do we continue to search for the ways in which we differ from one another? We can take an essential first step toward addressing this paradox by acknowledging the often conflicting stakes for individuals and groups in debates that center around genes, science, and race. Whose genes will be studied? For what purpose? And who has the authority to decide? These stakes are laid bare by Cassandra Medley in her play “Relativity,” which ran at the Magic Theater in San Francisco this spring, in a production directed by Edris Cooper-Anifowoshe. The play focuses on the inner conflict of a young scientist, an African American woman who tries unsuccessfully to straddle the opposing world views of her profession and her family. In “Relativity,” Kalima Davis is a postdoctoral fellow in a prestigious stem cell laboratory on the East Coast. Her impeccable academic pedigree distinguishes Kalima as a rising researcher, and she is one of the few women and African American scientists working in the field. Kalima is also the devoted daughter of the charismatic psychotherapist Claire Reid, who directs the “Leon Davis Foundation,” which Kalima's late father dedicated to the belief that neuropeptide melanin, found in “people of color,” enhances intelligence, athleticism, and emotional sensitivity. This theory also points to the lack of melanin among lighter skinned individuals as a cause of “white racism.” Kalima, who has inherited co-directorship of her father's foundation, is asked by her mother to offer “scientific proof” of the melanin theories and to discount the assertion that all groups are genetically similar. At issue in “Relativity” is the struggle over what constitutes a valid belief. Reid suggests to Kalima that “science is not the sole province of what the ‘West’ defines it to be,” and refers to Chinese acupuncture, Hindu Chakra, and tribal African shamanism as examples of legitimate “sciences.” But the power of Western science to trump other interpretations of lived experience has become all too clear in the genomic era. Truth is excavated from the human body, where genes emerge as the iconographic oracles of our past, present, and future. As Kalima recounts, “We can't get around it. DNA is fact.” Nonetheless, Kalima naively attempts to find a way to retain these opposing epistemologies. The futility of this dual position is made apparent by the arrival of Iris Preston, an African American senior scientist who has taken over as the new head of Kalima's lab. Preston, a highly vocal critic of Claire Reid and melanin theories, serves as a formidable apostle of the scientific method. Shortly after her arrival, Preston convenes members of her new lab to filter those seeking financial and other derivative rewards from the truly devoted, who are motivated solely by their “sense of wonder and amazement” and their desire to “cultivate what Einstein referred to as ‘holy curiosity.’” She makes plain that science, like all ideologies, demands consummate faith and unwavering piety. Kalima's struggle to claim her “true lineage” is much more than a simple choice between her biological mother (Reid) and her intellectual mentor (Preston). Her plight forces her to explore the meaning of justice. The contrast between melanin theories and genomic research, initially stark, blurs as it becomes increasingly clear that both Reid and Preston seek to use their “science” to redress race-related disparities. Citing a history of racism, including the historically well-documented Tuskegee Syphilis Study, Reid asks rhetorically whether genetics research will result in a “genetically modified” white upper class and a lower, dark-skinned “natural birth class”? At stake are issues of power and trust, and the question of whether new genetic technologies will close the gaps between groups or make them wider. Some postulate that the “new genetics” will render conventional notions of race obsolete. However, it is doubtful that such a color-blind utopia will be won through the sequencing of genes without a serious engagement with the differences that lie outside cell walls. A social infrastructure of inequality that mediates race through nutritional deficits, exposure to pollutants, and the use of the emergency room as the sole venue for healthcare will not be rehabilitated through gene therapy or pharmacogenomics. Preston seeks to use her stature in science to focus on the inequities within the academy. Encouraging Kalima to appear with her on a television program about stem-cell research, Preston urges her to imagine the milestone of “not just one, but two black women scientists, holding forth among the usual cadre of white males.” As one of the rare, highly scrutinized, “minority” scientists, Kalima embodies a “double jeopardy.” She must prove that she is worthy of her position—that she is as good, if not better, than her “white” peers—yet she must always be “remembering from whence she came.” When Dan, a white colleague in Kalima's lab who is also her boyfriend, hears that Preston has chosen Kalima to appear with her on television, he jealously accuses her of benefiting from preferential treatment. The play's depiction of this assertion of “reverse racism” questions the legitimate use of race in evaluating promotion and achievement in science. What is apparent is that race, while an ever-present subject, is often presented as the antithesis of conventional notions of a color-blind meritocracy. Left to linger is the critical question: does merely identifying differences among groups constitute an act of racism? Can statements of racial differences be neutral, unfettered by a relative hierarchy? Medley throws into stark relief the politics around race and gender in science. She illuminates how an individual's dilemma transcends the private realm; personal decisions are never truly “personal,” but are inherently public because they always have wider social repercussions. In the fateful confrontation between Kalima and her mother, Reid challenges her daughter to “grow up” and to risk her disapproval. A similar challenge can be issued to those who have inherited the “new genetics”—to vanquish the continuing paradox of reciting a “mantra of sameness” all the while searching for meaningful differences. In “Relativity,” Medley serves us a cautionary tale of the costs of our chronic ambivalence about the critical issues of race and justice in science. A good first step is to recognize that the search for meaning in human difference is inseparable from the struggle over the moral order in which we live. To underestimate the power of science to define our social agenda is to lose an opportunity to determine our future course. We only need to look to history as our proof. Sandra Soo-Jin Lee is a Senior Research Scholar in the Center for Biomedical Ethics and a Lecturer in the Department of Cultural and Social Anthropology at Stanford University. E-mail: [email protected]

0

PMC516793

CC BY

2021-01-05 08:21:15

no

PLoS Biol. 2004 Sep 14; 2(9):e263

utf-8

PLoS Biol

10.1371/journal.pbio.0020263

oa_comm

==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020285Community PageBioinformatics/Computational BiologyGenetics/Genomics/Gene TherapyEubacteriaHomo (Human)The Genome Assembly Archive: A New Public Resource Community PageSalzberg Steven L Church Deanna DiCuccio Michael Yaschenko Eugene Ostell James 9 2004 14 9 2004 14 9 2004 2 9 e285Copyright: © 2004 Salzberg et al.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.With the genome assembly archive, it is possible to examine the raw data that underlies the DNA sequence in any sequenced genome ==== Body Scientists have dedicated considerable effort to decoding the genomes of an ever-growing list of species, ranging from small viruses, whose genomes may be just a few thousand nucleotides in length, to large mammalian genomes, three billion nucleotides and larger. Many aspects of life science research have benefited by the accumulation of these data, but decoded genomes could be even more valuable if important information about the genome sequence, currently being lost, were preserved. Occasionally, questions arise about a specific position in the sequence—or a variant in the sequence is observed in a new sample. At times like these, it would be helpful to be able to go back to the experimental evidence that underlies the genome sequence at that position, to see if there is any ambiguity or uncertainty about the sequence. As things stand, that's almost impossible. To understand why this is the case, it is necessary to know a bit more about how a genome sequence is put together. Current sequencing technology can only generate 700–800 nucleotides at a time; genomes must therefore be shattered into many small fragments (in what is known as the “shotgun” approach), which are then sequenced. The sequences are assembled to generate a consensus sequence that, if all steps work perfectly, matches the original DNA molecule. Since the sequencing of Haemophilus influenzae in 1995 (Fleischmann et al. 1995), most bacterial and archaeal species have been sequenced by fragmenting the entire genome, sequencing the pieces, and assembling the result (the whole-genome shotgun, or WGS, strategy). In recent years, ever-larger sequencing projects have followed the WGS approach, requiring teams of computer experts and the use of increasingly sophisticated assembly algorithms in order to put together the huge number of sequence fragments. Without really being aware of it, the bioinformaticians who assemble genomes have for years been discarding the valuable information on how all of the individual sequence fragments align to the assembled chromosomes. This loss has gone largely unremarked because the scientific community has focused its attention primarily on the end product: the final genome sequence itself. It is only natural to regard the genome sequence, which is the basis for gene discovery and for functional understanding of the biology of the organism, as the primary result of a WGS project. In reality, though, a WGS project is an experiment in which large numbers of sequencing reactions are run, followed by a combination of computational work and additional sequencing to complete the genome. Three years ago, the Trace Archive (at The National Center for Biotechnology Information and The Wellcome Trust Genome Campus in Hinxton, United Kingdom) was developed to store the raw sequence data and to facilitate dissemination of this data, but currently there is no database that captures the alignment of these reads to the published genome sequence. Many scientists would be surprised to hear that genome assemblies are unavailable. One might infer that the assembly of a genome could be reconstructed from the genome sequence and the associated traces. However, aligning the traces to the genome will generally not reproduce the assembly, both because many of the traces will have alternate possible alignments and because, in some cases, parts of the assembly are manually refined based on additional experimental data. Furthermore, only a small number of large-scale centers have the computing hardware, software, and bioinformatics expertise to allow them to assemble a large genome. To bridge this gap, we have developed the Assembly Archive (http://www.ncbi.nlm.nih.gov/projects/assembly). The archive has been developed to store both an archival record of how a particular assembly was constructed and the alignments of any set of traces to a reference genome. Assemblies contained in this archive will be available in the GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html), DDBJ (http://www.ddbj.nig.ac.jp/), and EMBL (http://www.ebi.ac.uk/embl) databases, and all underlying traces are required to be deposited in the Trace Archive. The Assembly Archive's first entries are a set of seven closely related strains of Bacillus anthracis (the causative agent of anthrax), which have been sequenced as part of an effort to understand the detailed variation of that species. This includes the completed reference genome of the Ames strain, sequenced from a sample kept frozen since 1981, when it was originally isolated in West Texas (J. Ravel, personal communication). For the first time, the evidence behind each polymorphism in these assembled genomes will be directly accessible to the scientific community. Microbial Forensics Recently, heightened awareness of the threat of bioterrorism has spurred efforts to sequence genomes of multiple strains and isolates of a number of microbial pathogens, with the goal of cataloging all sequence differences between genomes. These efforts began with the study of the B. anthracis bacterium (the bacterium sent through the United States mail in late 2001) in order to determine if there were any differences between it and a reference laboratory sample (Read et al. 2002). This and subsequent studies have prompted many scientists to focus much greater attention on the assembly of a genome, and to regard the assembly rather than the genome as the object of greatest interest. In these forensic studies, we sequence whole genomes in order to discover every possible genetic difference between two bacteria or viruses. These genomes may differ in just one or two nucleotides out of millions that are identical; for example, the study referenced above uncovered just four single nucleotide polymorphisms (SNPs) in a chromosome of 5.23 million base pairs. The close similarity between the sequences forces us to consider all the facts behind each individual nucleotide that appears different. For studies that might be used as evidence in criminal investigations, it is essential to produce this information, and furthermore to quantify our confidence in each nucleotide in the genome. Regions of a genome with deep coverage are much more accurate than those with light coverage (i.e., regions with just one or two sequence reads). Figure 1 shows one of the interfaces in the Assembly Archive, covering a small region of the multiple alignment of sequences and traces to one of the newly deposited anthrax genomes. It also shows how it is possible to examine the evidence underlying a specific base in the DNA sequence. Figure 1 Snapshot of the Underlying Sequences and Traces from an Assembly of B. anthracis The consensus sequence shown across the top of the figure contains multiple sequences that validate each nucleotide in the window. Runs of a single base (monomer runs) are common causes of base-calling errors, because the peaks in the underlying trace data sometimes merge together. The sequence shown includes several monomer runs; several of the underlying traces are shown as well. For example, the run of six As at the far left of the figure is supported by several reads in which all six peaks are distinct, as well as other reads in which the six nucleotides appear as one broad peak. By examining data such as these, one can easily verify (or disprove) putative SNPs in this genome. Human SNP Research Human polymorphism studies (e.g., Sachidanandam et al. 2001) are a tremendously active and important area of research today. SNPs are directly implicated in a large number of diseases and inherited traits (Risch 2000, Chakravarti 2001). Within “haplotypes,” they describe individual variation for drug response (McLeod and Evans 2001) and provide a genetic framework for understanding disease phenotype (Hoehe 2003). In contrast with prokaryotic genomes, the human genome (as well as other animals, plants, and a broad range of eukaryotes) is diploid, and as a result many SNPs can be discovered within a single assembly, which contains the chromosomes representing the two parent organisms. SNPs can also be found through population studies in which the same locus is sampled from multiple individuals. In either case, the evidence for a SNP begins with the alignment of two different genomes. Despite the clear need for it, the original evidence for the genome itself—the assembly—is not available, and is not linked to the evidence in the Trace Archive. If it were available, many of the polymorphisms already reported could be validated, and many more SNPs might be discovered. Assemblies will also allow centers to better coordinate their gap-closing and finishing efforts, as has been recently noted (Schmutz et al. 2004). We hope that the availability of the Assembly Archive will encourage human genome sequencers, and sequencers of other genomes, to begin depositing their assemblies into this public resource, where it can be shared by all. Thanks to Martin Shumway, Mihai Pop, Steve Sherry, and David J. Lipman for help in defining the specifications and interface to the Assembly Archive, and for other valuable suggestions. SLS is supported in part by National Institutes of Health grant R01-LM06845. Steven L. Salzberg is with the Institute for Genomic Research (Rockville, Maryland, United States of America) and the Departments of Computer Science and Biology at Johns Hopkins University (Baltimore, Maryland, United States of America). Deanna Church, Michael DiCuccio, Eugene Yaschenko, and James Ostell are with the National Center for Biotechnology Information at the National Institutes of Health (Bethesda, Maryland, United States of America). E-mail: [email protected] (SLS). Abbreviations SNPsingle nucleotide polymorphism WGSwhole-genome shotgun ==== Refs References Chakravarti A Single nucleotide polymorphisms: To a future of genetic medicine Nature 2001 409 822 823 11236997 Fleischmann RD Adams MD White O Clayton RA Kirkness EF Whole-genome random sequencing and assembly of Haemophilus influenzae Rd Science 1995 269 496 512 7542800 Hoehe MR Haplotypes and the systematic analysis of genetic variation in genes and genomes Pharmacogenomics 2003 4 547 570 12943464 McLeod HL Evans WE Pharmacogenomics: Unlocking the human genome for better drug therapy Annu Rev Pharmacol Toxicol 2001 41 101 121 11264452 Read TD Salzberg SL Pop M Shumway M Umayam L Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis Science 2002 296 2028 2033 12004073 Risch NJ Searching for genetic determinants in the new millennium Nature 2000 405 847 856 10866211 Sachidanandam R Weissman D Schmidt SC Kakol JM Stein LD A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms Nature 2001 409 928 933 11237013 Schmutz J Wheeler J Grimwood J Dickson M Yang J Quality assessment of the human genome sequence Nature 2004 429 365 368 15164052

15367931

PMC516794

CC BY

2021-01-05 08:21:15

no

PLoS Biol. 2004 Sep 14; 2(9):e285

utf-8

PLoS Biol

10.1371/journal.pbio.0020285

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020297Journal ClubNeurosciencePsychologyHomo (Human)Bridging Psychology and Mathematics: Can the Brain Understand the Brain? Journal ClubSigman Mariano 9 2004 14 9 2004 14 9 2004 2 9 e297Copyright: © 2004 Mariano Sigman.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Mathematical measures of complexity shed light on why some concepts are inherently more difficult to learn than others ==== Body “It was not only difficult for him to understand that the generic term dog embraced so many unlike specimens of differing sizes and different forms; he was disturbed by the fact that a dog at three-fourteen (seen in profile) should have the same name as the dog at three-fifteen (seen from the front).…Without effort, he had learned English, French, Portuguese, Latin. I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details.” —Jorge Luis Borges, “Funes the Memorius” We are told scientists are divided into experimentalists and theoreticians. The dialectic description of the dynamics of science, with one tribe gathering data and collecting evidence and another tribe providing form to these observations, has striking examples that argue for the importance of synthesis. The 16th century revolution, which settled the way in which we see the sky today, is probably one of the best examples of how comparatively ineffective each of these tribes can be in isolation. Tycho Brahe, the exquisite observer, who built, calibrated, and refined instruments to see in the sky what no one else could, collected the evidence to prove a theory that Copernicus had already stated years before (in a book he dedicated to the Pope). It was only many years later that Galileo established the bridge between theory and observation; he understood the data in terms of the theory and thereby cemented the revolution. Copernicus's statements, showed Galileo, were not only figments of his imagination; they were an adequate description of the universe as he and Brahe had observed it. Since my first steps in biology, after a prompt departure from physics and mathematics, I have looked for such encounters between theory and experiment. I began studying the visual system and the series of fundamental works by Atneave (1954), Barlow (1960), and Atick (1992) on the relationship between our visual world and the way the brain processes it. Their research was based on a simple hypothesis: (1) the images that we see are highly redundant and (2) the optic nerve is a limited channel, thus the retina has to compress information. Compression, redundancy? How do such concepts relate to the biology of the brain? In the middle of the last century, working on the problem of communications, languages, codes, and channels, Claude Shannon proposed a very elegant theory that formalized intuitive ideas on the essence (and the limits) of communications (Weaver and Shannon 1949). One of its key aspects was that, depending on the code and on the input, channels are not used optimally and thus do not carry all the information they potentially could. When we compress (zip) a file, we are actually rewriting it in a more efficient (though not necessarily more intelligible) language in which we can store the same amount of information in less space. This compression has, of course, a limit (we cannot convey the universe in a single point), and the mere existence of an optimal code is central to Shannon's idea. Attneave was probably the first to think that these ideas could help unravel how the brain worked, and in a long series of works relating these ideas to experimental data, it was shown that the retina's business is mainly to get rid of the redundancies in the visual world. About four years ago, Jacob Feldman published a paper, similar in spirit, proposing a simple explanation for a long-standing problem in cognitive science about why some concepts are inherently more difficult to learn than others (Feldman 2000). An article whose first reference is to work carried out 50 years previously makes us suspect that an important gap is being filled. As in the previous experiments, Feldman borrowed a theory—he did not invent it—to explain longstanding and previously unexplained research in this area. Feldman's idea was based on a theory developed by Kolmogorov that established formal mathematical grounds to define and measure complexity. Kolmogorov's theory was focused on categories, which are just subsets of a universe, a bunch of exemplars that constitute a group. “Dogs” is a category in the universe of animals. Different statements can define the same category, for example, “big dogs” and “dogs that are not small” are the same group, and some information in the statement may be irrelevant because it does not aid in determining which elements belong or not. In the same way that Shannon spoke of a non-redundant code, Kolmogorov showed that categories could be defined with an optimal (non-redundant) statement. The length of this statement defines a measure of complexity termed Kolmogorov complexity. To visualize the intuitive nature of his theory it helps to do a thought experiment. Imagine a set of objects defined by, say, three features: form, color, and size. And imagine, for simplicity, that each feature is binary, that is, there are only two possible cases. Size is big or small, color is yellow or red, and shape can be either a triangle or a circle. This defines, of course, a universe of eight objects. We can now define categories within this universe: for example, all the circles (a category of four exemplars), or all the big and yellow objects (two exemplars), or all the triangles that are not red (again two) (see Figure 1). We can also define a category by enumeration, for example, the small red triangle, the big yellow circle, and the small yellow circle (three exemplars). Some rules (and thus the groups defined by these rules) are intuitively easier to define than others. “All the circles” is an easier statement to make (and probably to remember) than “small circles and yellow big objects.” This notion of difficulty is what Kolmogorov's theory formalized, stating that complexity was the length of the shortest definition (among all the possible definitions) of a given set. From this thought experiment, we can understand the logic of Feldman's paper, which showed that. Kolmogrov complexity is very closely related to our intuitive notion of conceptual difficulty. Feldman presented subjects with all possible categories (of a fixed number of exemplars) in different universes and showed that the critical parameter to rank the difficulty of a given subset was its Kolmogrov complexity. Moreover by explicitly presenting all the members and the nonmembers of a category to naïve subjects, he showed that we can spontaneously reduce a category to its minimal form and remember it without any explicit instruction. Thus, what Feldman found, following the original ideas of Shepard, was that our psychological measure of complexity—our difficulty in defining and remembering a category or concept—is also determined by the Kolmogorov complexity that describes it. Figure 1 Visualizing Kolmogorov´s Complexity Intuitive categories can be defined by short statements. The universe: circles and triangles, red and yellow, big and small (A). Examples of easy categories: red objects (B); triangles (C). Example of a difficult category: yellow circles and small red circles (D). This essay is, in a way, about how we avoid becoming Borges's character Funes, who could not understand repeated observations as exemplars of a common rule and thus could not synthesize and categorize. Simply, he could not think. Probably the most disappointing moment of Feldman's paper comes at the very end, where it deals with its (somehow unavoidable) recursive quest. Understanding why some concepts are difficult to learn may itself be difficult to learn. Modern mathematics, together with Kolmogorov complexity and information theory, has taught us another fundamental concept that may be relevant when trying to understand the logic of the mind. In a long series of paradoxes enumerated by Bertrand Russell, Kurt Goedel, and others, we learn that a formal system that looks at itself is bound to fail. At the very end of his paper, Feldman writes, “In a sense, this final conclusion [that psychological complexity is Boolean complexity] may seem negative: human conceptual difficulty reflects intrinsic mathematical complexity after all, rather than some idiosyncratic and uniquely human bias.” Who invented mathematics? The Martians? On the contrary, I believe this result supports a more naturalistic and less Platonic conception of mathematics. Formal ideas in mathematics are not arbitrary constructions of an arbitrary architecture; rather, they reflect the workings of the brain like a massive collective cognitive experiment. Mathematics does not only serve to help us understand biology; mathematics is biology. We are not less original if our thoughts resemble our mental constructions, we are just consistent. It is within this loop, this unavoidable recursion—mathematics understanding the logic of the brain—that we will have an opportunity to test, as some conspire, whether among all the wonders evolution has come out with, the ultimate might be a brain good enough to avoid the risk of understanding itself. Mariano Sigman is in the Cognitive Neuroimaging Research Unit of l'Institut National de la Santé et de la recherche Médicale, Orsay, France, and a fellow of the Human Frontiers Science Program. E-mail: [email protected] ==== Refs References Atick JJ Could information theory provide an ecological theory of sensory processing? Network 1992 3 213 251 Attneave F Informational aspects of visual perception Psychol Rev 1954 61 183 193 13167245 Barlow HB Thorpe WH Zangwill OL The coding of sensory messages Current problems in animal behaviour 1960 Cambridge Cambridge University Press 331 360 Feldman J Minimization of Boolean complexity in human concept learning Nature 2000 407 630 633 11034211 Weaver W Shannon CE The mathematical theory of communication 1949 Urbana (Illinois) University of Illinois Press 117

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PLoS Biol. 2004 Sep 14; 2(9):e297

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10.1371/journal.pbio.0020297

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020302Unsolved MysterySystems BiologyOtherNoneWhat Is Life—and How Do We Search for It in Other Worlds? Unsolved MysteryMcKay Chris P 9 2004 14 9 2004 14 9 2004 2 9 e302Copyright: © 2004 Chris P. McKay.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.If there is life on distant worlds, how would we go about finding it? ==== Body I need a “tricorder”—the convenient, hand-held device featured on Star Trek that can detect life forms even from orbit. Unfortunately, we don't have a clue how a tricorder might work, since life forms don't seem to have any observable property that distinguishes them from inanimate matter. Furthermore, we lack a definition of life that can guide a search for life outside Earth. How can we find what we can't define? An answer may lie in the observation that life uses a small, discrete set of organic molecules as basic building blocks. On the surface of Europa and in the subsurface of Mars, we can search for alien but analogous patterns in the organics. Life As We Know It The obvious diversity of life on Earth overlies a fundamental biochemical and genetic similarity. The three main polymers of biology—the nucleic acids, the proteins, and the polysaccarides—are built from 20 amino acids, five nucleotide bases, and a few sugars, respectively. Together with lipids and fatty acids, these are the main constituents of biomass: the hardware of life (Lehninger 1975, p 21). The DNA and RNA software of life is also common, indicating shared descent (Woese 1987). But with only one example of life—life on Earth—it is not all that surprising that we do not have a fundamental understanding of what life is. We don't know which features of Earth life are essential and which are just accidents of history. Our lack of data is reflected in our attempts to define life. Koshland (2002) lists seven features of life: (1) program (DNA), (2) improvisation (response to environment), (3) compartmentalization, (4) energy, (5) regeneration, (6) adaptability, and (7) seclusion (chemical control and selectivity). A simpler definition is that life is a material system that undergoes reproduction, mutation, and natural selection (McKay 1991). Cleland and Chyba (2002) have suggested that life might be like water, hard to define phenomenologically, but easy to define at the fundamental level. But life is like fire, not water—it is a process, not a pure substance. Such definitions are grist for philosophical discussion, but they neither inform biological research nor provide a basis for the search for life on other worlds. The simplest, but not the only, proof of life is to find something that is alive. There are only two properties that can determine if an object is alive: metabolism and motion. (Metabolism is used here to include an organism's life functions, biomass increase, and reproduction.) All living things require some level of metabolism to remain viable against entropy. Movement (either microscopic or macroscopic) in response to stimuli or in the presence of food can be a convincing indicator of a living thing. But both metabolism (fire) and motion (wind) occur in nature in the absence of biology. The practical approach to the search for life is to determine what life needs. The simplest list is probably: energy, carbon, liquid water, and a few other elements such as nitrogen, sulfur, and phosphorus (McKay 1991). Life requires energy to maintain itself against entropy, as does any self-organizing open system. In the memorable words of Erwin Schrödinger (1945), “It feeds on negative entropy.” On Earth, the vast majority of life forms ultimately derive their energy from sunlight. The only other source of primary productivity known is chemical energy, and there are only two ecosystems known, both methanogen-based (Stevens and McKinley 1995; Chapelle et al. 2002), that rely exclusively on chemical energy (that is, they do not use sunlight or its product, oxygen). Photosynthetic organisms can use sunlight at levels below the level of sunlight at the orbit of Pluto (Ravens et al. 2000); therefore, energy is not the limitation for life. Carbon, nitrogen, sulfur, and phosphorus are the elements of life, and they are abundant in the Solar System. Indeed, the Sun and the outer Solar System have more than 10,000 times the carbon content of the bulk of Earth (McKay 1991). When we scan the other worlds of our Solar System, the missing ecological ingredient for life is liquid water. It makes sense, then, that the search for liquid water is currently the first step in the search for life on other worlds. The presence of liquid water is a powerful indication that the ecological prerequisites for life are satisfied. Orbital images, such as the canyon in Figure 1, show clear evidence of the stable and repeated, if not persistent, flow of a low-viscosity fluid on Mars at certain times in its past history. The fluid was probably water, but the images could also suggest wind, ice, lava, even carbon dioxide or sulfur dioxide. Recently, results from the Mars Exploration Rover missions have shown that this liquid carried salts and precipitated hematite in concretions. The case for water, we could say, is tight. Figure 1 Water on Another World A Mars Global Surveyor image showing Nanedi Vallis in the Xanthe Terra region of Mars. The image covers an area 9.8 km ×18.5 km; the canyon is about 2.5 km wide. This image is the best evidence we have of liquid water anywhere outside the Earth. Photo credit: NASA/Malin Space Sciences. On Jupiter's moon Europa, the cracks and icebergs on the surface of the ice indicate water beneath the ice, but not necessarily at the present time. Present water on Europa is indicated by the magnetic disturbance Europa makes as it moves through Jupiter's magnetic field, not unlike the way coins in the pocket of a passenger will set off an airport metal detector. Europa has a large conductor, and this is most likely a global, salty layer of water. Viking on Mars: Been There, Tried That The Viking missions to Mars in the late 1970s were the first (and as yet, the only) search for life outside Earth. Each Viking conducted three incubation experiments to detect the presence of metabolism in the Martian soil. Each lander also carried a sophisticated Gas Chromatograph Mass Spectrometer for characterizing organic molecules. The results were unexpected (Klein 1978, 1999). There was a detectable reaction in two of the incubation experiments. In the “Gas Exchange” experiment, a burst of oxygen was released when the soil was exposed to water. The “Labeled Release” experiment showed that organic material was consumed, and that carbon dioxide was released concomitantly. In the Labeled Release experiment, this reaction ceased if the soil was first heated to sterilizing temperatures, but the reaction of the Gas Exchange Experiment persisted. If considered alone, the Labeled Release results would be a plausible indication for life on Mars. However, the Gas Chromatograph Mass Spectrometer did not detect the presence of any organic molecules in the soil at level of one part per billion (Biemann 1979). It is difficult to imagine life without associated organic material, and this is the main argument against a biological interpretation of the Viking results (Klein 1999; but cf. Levin and Straat 1981). It is also unlikely that the oxygen release in the Gas Exchange experiment had a biological explanation, because the reaction was so rapid and persisted after heating. It is generally thought that the reactivity observed by the Viking biology experiments was caused by one or more inorganic oxidants present in the soil, and was ultimately produced by ultraviolet light in the atmosphere. Consistent with the apparently negative results of the Viking biology experiments, the surface of Mars also appears to be too dry for life. Indeed, conditions are such that liquid water is rare and transient, if it occurs at all (e.g., Hecht 2002). It's Life, Jim, but Not As We Know It Table 1 shows a categorization of life as we have observed it. Using this diagram, we can speculate about how life might be different on Mars or Europa. At the bottom of the table, life is composed of matter—a reasonable assumption for now. Carbon and liquid water are the next level; this makes Mars and Europa likely candidates, because they have carbon and have, or have had, liquid water. Other worlds may have a different chemical baseline for life. The usual speculation in this area is that the presence of ammonia and silicon, rather than water and carbon, might be preconditions for life on other planets. Such speculation has yet to lead to any specific suggestions for experiments, or to new ways to search for such life, but this may just reflect a failure of human imagination rather than a fundamental limitation on the nature of life. Table 1 A Categorization of Structures That Comprise Terrestrial Life Life on Mars is also likely to be the same at the top of the table: at the ecological level. Primary production in a Martian ecosystem is likely to be phototrophic, using carbon dioxide and water. Heterotrophs are likely to be present to consume the phototrophs and in turn to be consumed by predators. Darwinian evolution would result in many of the same patterns we see in ecosystems on Earth. While it may be similar at the top (ecological) and bottom (chemical) levels, life on Mars could be quite alien in the middle, in the realm of biochemistry. Pace (2001) has argued that alien biochemistry will turn out to be the same as biochemistry on Earth, because there is one best way to do things and that natural selection will ensure that life everywhere discovers that way. Only observation will tell if there is one possible biochemistry, or many. Future missions to Mars might find microfossils in sedimentary rocks such as those at Meridiani Planum. Microbes don't readily form convincing fossils; the one exception may be the strings of magnetite formed by magnetotactic bacteria (Friedmann et al. 2001). As interesting as fossils might be, we could not be sure that a fossil found on Mars was not merely another example of Earth life. We know that rocks have come to Earth from Mars, and it is possible that such rocks could have carried life between the planets (Mileikowsky et al. 2000; Weiss et al. 2000). Finding fossil evidence for life on Mars does not demonstrate a second genesis in our Solar System. Finding a Way to Search for Alien Life If we were to find organic material in the subsurface of Mars or on the ice of Europa, how could we determine whether it was the product of a system of biology or merely abiotic, organic material from meteorites or photochemistry? If this life were in fact related to Earth life, this should be easy to determine. We now have very sensitive methods, such as PCR and fluorescent antibody markers, for detecting life like us. This case would be the simplest to determine, but it would also be the least interesting. If the life turned out to be truly alien, then the probes specific to our biology would be unlikely to work. What, then, could we do to determine a biological origin? The question is open and possibly urgent. On space missions already being planned, we may have the opportunity to analyze the remains of alien organics on the surface of Europa or frozen below ground on Mars. The instruments that will make this analysis must be designed in the next couple of years. One approach appears promising. I call it the “Lego Principle.” It is based on the patterns of the molecules of life. Biological processes, in contrast to abiotic mechanisms, do not make use of the range of possible organic molecules. Instead, biology is built from a selected set. Thus, organic molecules that are chemically very similar to each other may have widely different concentrations in a sample of biological organics. An example of this on Earth is the 20 amino acids used in proteins and the use of the left-handed version of these amino acids. The selectivity of biological processes is shown schematically in Figure 2 by the distribution of spikes in contrast to a smooth, nonbiological distribution. General arguments of thermodynamic efficiency and specificity of enzymatic reactions suggest that this selectivity is required for biological function and is a general result of natural selection. Different life forms are likely to have different patterns, and at the very least we might find the mirror symmetry of life on Earth, with d- instead of l-amino acids. Figure 2 Comparison of Biogenic with Nonbiogenic Distributions of Organic Material Nonbiological processes produce smooth distributions of organic material, illustrated here by the curve. Biology, in contrast selects and uses only a few distinct molecules, shown here as spikes (e.g., the 20 l-amino acids on Earth). Analysis of a sample of organic material from Mars or Europa may indicate a biological origin if it shows such selectivity. This approach has immediate practical benefit in the search for biochemistry in the Solar System. Samples of organic material collected from Mars and Europa can be easily tested for the prevalence of one chirality of amino acid over the other. More generally, a complete analysis of the relative concentration of different types of organic molecules might reveal a pattern that is biological even if that pattern does not involve any of the familiar biomolecules. Interestingly, if a sample of organics from Mars or Europa shows a preponderance of D-amino acids, this would be evidence of life, and at the same time would show that this life was distinct from Earth life. This same conclusion would apply to any clearly biological pattern that was distinct from that of Earth life. Organic material of biological origin will eventually lose its distinctive pattern when exposed to heat and other types of radiation, (examples of this include the thermal racemization of amino acids), but at the low temperatures in the Martian permafrost, calculations suggest that there has been no thermal alteration (Kanavarioti and Mancinelli 1990). An interesting question, as yet unanswered, is how long organic material frozen into the surface ice of Europa would retain a biological signature in the strong radiation environment. On Europa, the organic material for our tests might be collected right from the dark regions on the surface. On Mars, there is ice-rich ground in the cratered southern polar regions (Feldmann et al. 2002), which presumably overlies deeper, older ice. The surprise discovery of strong magnetic fields in the southern hemisphere of Mars (Acuña et al. 1999; Connerney et al. 1999) indicates that the area may be the oldest undisturbed permafrost on that planet. Like the mammoths extracted from the ice in Siberia, any Martian microbes found in this ice would be dead, but their biochemistry would be preserved. From these biological remains, it would then be possible to determine the biochemical composition of, and the phylogenetic relationship between, Earth life and Martian life. We may then have, for the first time, a second example of life. Chris P. McKay is with the NASA Ames Research Center. E-mail: [email protected] ==== Refs References Acuña MH Connerney JEP Ness NF Lin RP Mitchell D Global distribution of crustal magnetism discovered by the Mars Global Surveyor MAG/ER experiment Science 1999 284 790 793 10221908 Biemann K The implications and limitations of the findings of the Viking Organic Analysis Experiment J Mol Evol 1979 14 65 70 522160 Chapelle FH O'Neil K Bradley PM Methé BA Ciufo SA A hydrogen-based subsurface microbial community dominated by methanogens Nature 2002 415 312 315 11797006 Cleland CE Chyba CF Defining life Orig Life Evol Biosph 2002 32 387 393 12458739 Connerney JEP Acuna MH Wasilewski P Ness NF Reme H Magnetic lineations in the ancient crust of Mars Science 1999 284 794 798 10221909 Feldman WC Boynton WV Tokar RL Prettyman TH Gasnault O Global distribution of neutrons from Mars: Results from Mars Odyssey Science 2002 297 75 78 12040088 Friedmann EI Wierzchos J Ascaso C Winklhofer M Chains of magnetite crystals in the meteorite ALH84001: Evidence of biological origin Proc Natl Acad Sci U S A 2001 98 2176 2181 11226212 Hecht MH Metastability of liquid water on Mars Icarus 2002 156 373 386 Kanavarioti A Mancinelli RL Could organic matter have been preserved on Mars for 3.5 billion years? Icarus 1990 84 196 202 11538399 Klein HP The Viking biological experiments on Mars Icarus 1978 34 666 674 Klein HP Did Viking discover life on Mars? Orig Life Evol Biosph 1999 29 625 631 10666745 Koshland DE The seven pillars of life Science 2002 295 2215 2216 11910092 Lehninger AL Biochemistry 1975 >New York Worth 1104 Levin GV Straat PA A search for nonbiological explanation of the Viking labeled release life detection experiment Icarus 1981 45 494 516 McKay CP Urey Prize lecture: Planetary evolution and the origin of life Icarus 1991 91 92 100 McKay CP The search for life on Mars Orig Life Evol Biosph 1997 27 263 289 9150577 Mileikowsky C Cucinotta FA Wilson JW Gladman B Horneck G Natural transfer of viable microbes in space Icarus 2000 145 391 427 11543506 Pace N The universal nature of biochemistry Proc Natl Acad Sci U S A 2001 98 805 808 11158550 Raven JA Kübler JE Beardall J Put out the light, and then put out the light J Mar Biol Ass (UK) 2000 80 1 25 Schrödinger E What is life? 1945 Cambridge Cambridge University Press 91 Stevens TO McKinley JP Lithoautotrophic microbial ecosystems in deep basalt aquifers Science 1995 270 450 454 Ultee A Souvatzi N Maniadi K König H Identification of the culturable and nonculturable bacterial population in ground water of a municipal water supply in Germany J Appl Microbiol 2004 96 560 568 14962136 Weiss BP Kirschvink JL Baudenbacher FJ Vali H Peters NT A low temperature transfer of ALH84001 from Mars to Earth Science 2000 290 791 795 11052940 Woese CR Bacterial evolution Microbiol Rev 1987 51 221 271 2439888

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PLoS Biol. 2004 Sep 14; 2(9):e302

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PLoS Biol

10.1371/journal.pbio.0020302

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020307PrimerEvolutionImmunologyVirologyVirusesHomo (Human)Adaptation and Immunity PrimerHolmes Eddie C 9 2004 14 9 2004 14 9 2004 2 9 e307Copyright: © 2004 Eddie C. Holmes.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Ancient Adaptive Evolution of the Primate Antiviral DNA-Editing Enzyme APOBEC3G The ongoing battle between hosts and pathogens has long been of interest to evolutionary biologists. ==== Body The ongoing battle between hosts and pathogens has long been of interest to evolutionary biologists. Because hosts and pathogens act as environments for each other, their intertwined struggle for existence is both continual and rapid. At the molecular level, this cycle of environmental change and evolutionary response means that mutations are continually being tried out by natural selection. It is therefore little wonder that the host and pathogen genes that control infection and immunity frequently show high levels of genetic diversity and present some of the best examples of positive selection (adaptive evolution) reported to date (Yang and Bielawski 2000). In particular, rates of nonsynonymous substitution per site (resulting in an amino acid change; dN) often greatly exceed those of synonymous substitution per site (silent change; dS), as expected if most mutations are fixed because they increase fitness (Figure 1). Figure 1 Measuring Selection Pressures by Comparing the Ratio of Nonsynonymous to Synonymous Substitutions Per Site (A) Classification of substitutions. Nonsynonymous substitutions (red) are those that change the amino acid sequence of the protein encoded by the gene, while the degeneracy of the genetic code ensures that synonymous substitutions (yellow) result in the same amino acid sequence. (B) Calculation of dN/dS. By assuming that synonymous mutations are neutral and fixed by random genetic drift, it is possible to determine the mode of selection acting on nonsynonymous mutations. If all nonsynonymous substitutions were neutral, then their rate of occurrence per site (dN) would be the same as that of synonymous substitutions per site (dS), so that dN/dS equals one. A lower ratio of nonsynonymous to synonymous substitutions per site (dN/dS < 1) means that some proportion of the nonsynonymous mutations are deleterious and removed by purifying selection. Conversely, positive selection fixes advantageous nonsynonymous mutations faster than genetic drift fixes synonymous mutations (dN/dS > 1), although this is usually restricted to a small proportion of amino acid sites within any gene. In the hypothetical example of five gene sequences shown here, with dS given above the diagonal and dN below the diagonal, there is no evidence for positive selection because mean dN/dS the (0.577) is less than one. At the host level, most studies of the selection pressures acting on immune system genes have concentrated on genes implicated in the adaptive immune response against microbial pathogens, particularly those producing antibodies (Sitnikova and Nei 1998; Sumiyama et al. 2002), or genes encoding reconnaissance molecules known as the major histocompatibility complex (MHC), which control the action of T-cells (Hughes and Nei 1988; Yeager and Hughes 1999) (Box 1). As its name suggests, the role of the adaptive immune response is to stimulate and ‘memorise’ immunity to specific pathogens. As microbial pathogens such as viruses are both abundant and rapidly evolving, positive selection on components of the adaptive immune response is often very strong (Yeager and Hughes 1999). Far less attention has been directed toward the less specific innate (‘nonadaptive’) immune response, even though this response requires a wide array of genes and acts as the front line of immune defence (Box 1). Would we expect the same strength of positive selection on a generalized pathogen control system? This is a question of fundamental importance because the luxury of adaptive immunity is not available to most organisms, having probably evolved along with the vertebrates (Bartl et al. 1994), whereas the more widespread innate immune system is often depicted as a primitive characteristic. Molecular Evolution of the Innate Immune System The genes involved in innate immunity have recently come under the molecular evolutionists' gaze. One important group are the defensins, a large class of short antimicrobial peptides that constitute an effective immune response team in organisms as diverse as plants and primates (Boman 1995). Because defensins are cationic (positively charged), they are able to interact with negatively charged molecules on the surface of microbes and permeate their membranes. Sequence analyses of defensins and similar antimicrobial peptides have revealed the telltale signatures of positive selection, with dN greater than dS in many comparisons (Hughes 1999; Duda et al. 2002; Maxwell et al. 2003). Other genes of the innate immune system also seem to be subject to powerful positive selection. One dramatic example described in this issue of PLoS Biology is the APOBEC3G gene of primates (Sawyer et al. 2004). This case is especially striking because rather than killing pathogens through protein or cellular interactions, like most immune genes, APOBEC3G works by manipulating the genome sequence of the invading microbe. The genomes of primates contain a family of nine APOBEC genes that encode enzymes involved in the editing of RNA and/or DNA through the deamination of cytosine (C), so that this nucleotide mutates to uracil (U). This is essential for various aspects of cellular function. APOBEC1, for example, is involved in the C→U editing of apolipoprotein B mRNA (therein christening the family), while another family member, the activation-induced deaminase, has a vital role in adaptive immunity in that it assists in the diversification of antibodies. Two more enzymes, APOBEC3G and APOBEC3F, form part of the innate immune system; they function as antiviral agents and are being intensively studied in the context of infection with the human immunodeficiency virus (HIV), the cause of AIDS. In particular, APOBEC3G targets the reverse transcription step of the HIV life cycle, in which the viral genomic RNA is converted into proviral DNA, which is then integrated into the host genome (Mangeat et al. 2003). APOBEC3G-induced deamination at this stage results in monotonous guanine-to-adenine (G→A) nucleotide changes, a phenomenon called G→A hypermutation that had long been noted by HIV researchers without a clear understanding of its cause. We now know that G→A hypermutation is part of the innate immune response to retroviral infections. Although there is still some debate over exactly how APOBEC3G leads to viral eradication, the most likely scenario is that G3A hypermutation results in the generation and incorporation of a multitude of deleterious mutations that fatally disrupt viral functions. This strategy is likely to work well for retroviruses like HIV because their genomes are so compact that individual sequence regions often perform multiple functions. Under these cramped conditions, most mutations are likely to severely disrupt some aspect of viral function and thereby reduce fitness (Holmes 2003). Indeed, it has been estimated that the deleterious mutation rate in viruses that replicate using RNA polymerases (either reverse transcriptase in the case of retroviruses or RNA-dependent RNA polymerase for other RNA viruses) is on the order of one error per replication cycle, so that many of the viral progeny produced by replication are defective (Elena and Moya 1999). HIV is normally able to overcome this burden of deleterious mutation because of its remarkable reproductive power; each day, on the order of 1010 virions are produced in a single infected individual (Perelson et al. 1996), so that enough fit and able recruits will make it through to the next generation. Treating RNA Virus Infections Through Lethal Mutagenesis The high mutation rates of RNA viruses mean that adaptively useful genetic variation is produced frequently. The rub, however, is that fitness-enhancing mutations are a small minority, and the preponderance of deleterious mutations means that RNA viruses live on the edge of survival (Domingo 2000). By increasing the rate at which deleterious mutations appear, APOBEC3G pushes viruses over this edge, causing a form of ‘lethal mutagenesis’ that results in their destruction; the rate of mutation becomes so high that no genome can reproduce itself faithfully, and the population crashes. Intriguingly, researchers designing new antiviral drugs have also begun to realise that forcing viruses into this sort of ‘error catastrophe’ might be an effective way to treat them (Figure 2). There are a growing number of studies in which mutagens, such as ribavirin and 5-fluorouracil, are applied to viral infections in vitro and in vivo, including HIV, in the hope that these will induce so many deleterious mutations that the virus suffers an error catastrophe and is cleared (Loeb et al. 1999; Sierra et al. 2000; Crotty et al. 2001; Ruiz-Jarabo et al. 2003). The results produced to date are highly encouraging, particularly when these error-inducing drugs are combined with more conventional treatment strategies that aim to reduce the rate of viral replication (Pariente et al. 2001). The discovery that a natural antiviral agent, APOBEC3G, probably works in much the same way should provide even more encouragement. Figure 2 Lethal Mutagenesis As a Means of Controlling RNA Virus Infections (A) In a viral population prior to the application of mutagens, the mean error rate (white) is on the order of one per genome per replication (mutations marked by asterisks). (B) If a mutagen such as ribavirin is then applied to an infected patient, the mean error rate of the virus (black) is increased so that the population crosses a threshold of ‘error catastrophe’; after this point fitness declines dramatically and the population crashes. This drug-induced lethal mutagenesis seems to work more efficiently when it is used in combination with drugs that reduce the rate of viral replication. Sadly, however, the pathogens have fought back. The anti-HIV properties of APOBEC3G were discovered because most viral strains escape its neutralising properties. Lentiviruses like HIV possess a gene that encodes an protein called Vif (‘viral infectivity factor’) that counters APOBEC3G (Sheehy et al. 2002). Hence, it is probably only in naturally occurring Vif-defective mutants that APOBEC3G is effective against HIV. Furthermore, because positive selection on APOBEC3G has operated for at least 30 million years and lentiviruses in general, and HIV in particular, are likely to be more recently evolved than this, it is clear that a broad range of retroviral pathogens have been responsible for the adaptive evolution of this particular immune gene (Sawyer et al. 2004). Given the frequency with which the remnants of past retroviral infections are found in the mammalian genome (Smit 1999), in the form of usually defunct endogenous retroviruses (Box 1), it is likely that our genomes are continually bombarded with retroviruses like HIV but that the majority are cleared by innate immune mechanisms like APOBEC3G. It is possible that the retroviruses that successfully infect us are those, like HIV, that have managed to evolve strategies to avoid the destructive capacities of APOBEC3G. The intense selective pressure on the defensins and APOBEC3G illustrates that although the innate immune response is generalist in its action, it is as highly and intricately evolved as its better-studied ally, the adaptive immune system. Rather than being an evolutionary remnant, it is a dynamic and continually adapting system. Less clear is whether other host proteins act in the same manner as APOBEC3G. In particular, the most common and destructive pathogens faced by humans and other mammals are RNA viruses, such as influenza A, yellow fever, and hepatitis C. In most cases, our ability to survive these viral infections is simply a combination of good luck and good breeding; with the right combination of MHC alleles, itself a function of population history and what we by chance inherit from our parents, some individuals may be more able to fight off viral infections than others. The ubiquity of RNA viruses hints that our genomes might also contain an innate, yet highly adapted, defence system that targets this abundant class of pathogens by manipulating their mutation rate. Although utilizing lethal mutagenesis might one day be an important way to design new drugs against a variety of viral pathogens, it would come as no surprise if nature got there first. Box 1. Glossary Adaptive immune system. The pathogen-specific part of the vertebrate immune system. It is comprised of two major arms, antibodies (the humoral response) and T-cells (the cellular response), both of which lay down an immunological memory for future defence. Innate immune system. The nonspecific part of the vertebrate immune system. It has a wide variety of components, ranging from lysozymes in saliva to cytokines, defensins, interferons, and natural killer cells in a variety of tissues. Endogenous retroviruses. The (usually) dead remnants of functional retroviruses that are now passed on through the germ line like normal genes. It has been estimated that approximately 5% of the human genome is composed of endogenous retroviruses. Eddie C. Holmes is in the Department of Zoology at the University of Oxford, Oxford, United Kingdom. E-mail: [email protected] Abbreviations Ccytosine dNrate of nonsynonymous substitution per site dSrate of synonymous substitution per site G→Aguanine-to-adenine HIVhuman immunodeficiency virus MHCmajor histocompatibility complex Uuracil ==== Refs References Bartl S Baltimore D Weissman IL Molecular evolution of the vertebrate immune system Proc Natl Acad Sci U S A 1994 91 10769 10770 7971960 Boman HG Peptide antibiotics and their role in innate immunity Annu Rev Immunol 1995 13 61 92 7612236 Crotty S Cameron CE Andino R RNA virus error catastrophe: Direct test by using ribavirin Proc Natl Acad Sci U S A 2001 98 6895 6900 11371613 Domingo E Viruses on the edge of adaptation Virology 2000 270 251 253 10792982 Duda TF Vanhoye D Nicholas P Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides Mol Biol Evol 2002 19 858 864 12032242 Elena SF Moya A Rate of deleterious mutation and the distribution of its effects on fitness in vesicular stomatitis virus J Evol Biol 1999 12 1078 1088 Holmes EC Error thresholds and the constraints to RNA virus evolution Trends Microbiol 2003 11 543 546 14659685 Hughes AL Evolutionary diversification of the mammalian defensins Cell Mol Life Sci 1999 56 94 103 11213266 Hughes AL Nei M Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection Nature 1988 335 367 370 Loeb LA Essigmann JM Kazazi F Zhang J Rose KD Lethal mutagenesis of HIV with mutagenic nucleoside analogs Proc Natl Acad Sci U S A 1999 96 1492 1497 9990051 Mangeat B Turelli P Caron G Friedli M Perrin L Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts Nature 2003 424 99 103 12808466 Maxwell AI Morrison GM Dorin JR Rapid sequence divergence in mammalian ß-defensins by adaptive evolution Mol Immunol 2003 40 413 421 14568387 Pariente N Sierra S Lowenstein PR Domingo E Efficient virus extinction by combinations of a mutagen and antiviral inhibitors J Virol 2001 75 9723 9730 11559805 Perelson AS Neumann AU Markowitz M Leonard JM Ho DD HIV-1 dynamics in vivo: Virion clearance rate, infected cell lifespan, and viral generation time Science 1996 271 1582 1586 8599114 Ruiz-Jarabo CM Ly C Domingo E de la Torre JC Lethal mutagenesis of the prototypic arenavirus lymphocytic choriomeningitis (LCMV) Virology 2003 308 37 47 12706088 Sawyer SL Emerman M Malik HS Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G PLoS Biol 2004 2 e275 15269786 Sheehy AM Gaddis NC Choi JD Malim MH Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein Nature 2002 418 646 650 12167863 Sierra S Dávila M Lowenstein PR Domingo E Response of foot-and-mouth disease virus to increased mutagenesis. Influence of viral load and fitness in loss of infectivity J Virol 2000 74 8316 8323 10954530 Sitnikova T Nei M Evolution of immunoglobulin kappa chain variable region genes in vertebrates Mol Biol Evol 1998 15 50 60 9491604 Smit AF Interspersed repeats and other mementos of transposable elements in mammalian genomes Curr Opin Genet Dev 1999 9 657 663 10607616 Sumiyama K Saitou N Ueda S Adaptive evolution of the IgA hinge region in primates Mol Biol Evol 2002 19 1093 1099 12082128 Yang Z Bielawski JP Statistical methods for detecting molecular adaptation Trends Ecol Evol 2000 15 496 502 11114436 Yeager M Hughes AL Evolution of the mammalian MHC: Natural selection, recombination, and convergent evolution Immunol Rev 1999 167 45 58 10319250

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PLoS Biol. 2004 Sep 14; 2(9):e307

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PLoS Biol

10.1371/journal.pbio.0020307

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020310FeatureEcologyScience PolicyAnimalsPlantsThe Conservation Business FeatureNicholls Henry 9 2004 14 9 2004 14 9 2004 2 9 e310Copyright: © 2004 Henry Nicholls.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Direct payments to local communities to conserve wildlife could prove effective but is biodiversity a commodity that can be bought and sold? ==== Body The language of conservation is changing: protecting biodiversity is no longer just about ethics and aesthetics; the latest buzzwords are commodities and consumers. Traditionally, conservation initiatives have talked up the benefits they will bring to the global community—saving species, habitats, ecosystems, and ultimately the planet. But conservation also has its costs, and these are usually borne by local people prevented from exploiting the resources around them in other ways. It is unfair to expect a localised minority to pick up costs that ultimately benefit a dispersed majority, argue conservation biologists. There has to be more money made available by concerned individuals, non-governmental organisations, national governments, and international bodies, and there need to be better ways to spend this money if conservation is to be effective, they say. Biodiversity is a commodity that can be bought and sold. We are consumers and must pay. Costs and Benefits Kenya boasts one of the world's most spectacular networks of national parks and reserves covering around 60,000 km2 of the country (Figure 1). But devoting such a vast area to conservation has its drawbacks. It has been estimated that were this land developed it would be worth around $270 million to the Kenyan people every year. Similarly, two national parks in Madagascar are estimated to have reduced the annual income of local villagers by around 10%. Of course, protected areas do bring some benefits to neighbouring communities, most notably through tourism. But in many cases the rewards are not great, they are rarely distributed evenly among individuals, and do not necessarily outweigh the costs. Figure 1 The Masai Mara National Park in Kenya Courtesy of Charlotte Stirling. ‘The costs of conservation fall disproportionately on local people, whereas the benefits are dispersed,’ says Andrew Balmford, a conservation biologist at the University of Cambridge in the United Kingdom. National and global communities stand to benefit from conservation of tropical biodiversity, but they must pay if they want to realise that benefit, he says. Conservation expenditure in the developed world is only about a third of what is needed for effective protection of 15% of the earth's terrestrial habitats, an area just large enough to preserve a representative sample of species, habitats, and ecosystems in the medium to long term (Balmford et al. 2003). The developed world must make up this funding shortfall, argues Balmford. What's more, there need to be smarter ways to spend the money that's available, he says. Conservation by Distraction In recent years, many funding bodies have taken an indirect approach to conservation, investing in projects that encourage people to take up alternative practices that are compatible with conservation rather than investing in conservation itself. Perhaps the best example of this ‘conservation by distraction’ is ploughing money into community-based ecotourism projects. Such initiatives aim to bring the benefits of tourism to local people, thereby encouraging them to preserve the biodiversity they have. It's an attractive idea. In the mid 1990s, the United States Agency for International Development was investing more than $2 billion a year in 105 conservation projects with an ecotourism component. Similarly, between 1988 and 2003, the World Bank funded 55 development projects that supported protected areas in Africa, 32 of which placed an emphasis on ecotourism. However, an absence of quantitative data and analysis has made it hard to judge whether these projects actually achieve their dual purpose of preserving biodiversity and simultaneously reducing rural poverty. ‘Much of the information about community-based ecotourism is anecdotal and subjective,’ says Agnes Kiss of the Environment and Social Development Unit at the World Bank. The real contribution of these initiatives to biodiversity conservation is debatable, she says. ‘Many community-based ecotourism projects cited as success stories actually involve little change in existing local land- and resource-use practices, provide only modest supplement to local livelihoods, and remain dependent on external support for long periods, if not indefinitely’ (Kiss 2004). For example, communities involved in the Infierno Community Ecotourism Project in Peru have received nearly $120,000 from their share in a tourist lodge and wages for providing services to visitors. This may have increased the income for a minority that are lodge employees, but only one family, whose adult members were all employed by the lodge, could afford to live solely on tourism. In the community as a whole, the average annual income from tourism was only $735 compared with nearly $2,000 earned elsewhere. Most of the community was still heavily dependent on other activities, and most of those activities are somewhat disruptive of conservation goals, says Kiss. Johan du Toit of the Mammal Research Institute at the University of Pretoria in South Africa is also critical of this kind of indirect approach to conservation. At the heart of the argument for community-based ecotourism is the idea of the ‘ecologically noble savage’, he says—the notion that those living closest to nature will know what's best for it. ‘It's a wonderful idea, but it just doesn't work. Nowhere in the history of evolution has sustainability ever been naturally selected for,’ says du Toit. ‘The AK47 automatic assault rifle has replaced the bow and arrow.…Every individual in a rural community that's out hunting will shoot what he sees when he sees it, because if he doesn't somebody else will.’ Nowhere is this problem more evident than in the ecotourist paradise of the Galápagos Islands (Figure 2), where a small minority of fishermen is coming into conflict with conservation aims with increasing regularity (Box 1). ‘Things are going down very quickly,’ says one Galápagos guide. ‘The iceberg is starting to tip over, and we are going to lose everything.’ If it still pays locals to exploit the environment rather than take part in one of the world's most buoyant ecotourism industries, it is clear that ecotourism alone cannot solve the world's conservation problems. Many think that ‘direct payment’ could be a useful tool. ‘Direct payment, very boldly speaking, is paying people in rural areas not to bugger up their environment,’ says du Toit. ‘It's just like if we want exclusive artworks to be looked after in the Louvre Gallery in Paris. Somebody's got to pay for it,’ he says. ‘You can't expect the Parisians who live in that arrondissement to cover the costs.’ Figure 2 Ecotourist Paradise in the Galápagos Courtesy of Catriona MacCallum. You Get What You Pay for—You Should Pay for What You Want to Get For people living in developing countries, where most of the world's biodiversity exists, the short-term rewards of exploiting these natural resources are significant. Replacing indirect conservation measures, such as community-based ecotourism, with payments directly into the pockets of local people could turn out to be a much more effective way to stem this exploitation, argues Paul Ferraro, an economist at Georgia State University in Atlanta (Ferraro and Kiss 2002). It could also bring far greater development benefits than indirect financial support, he says (Box 2). An additional spin-off is that direct payments force conservation biologists to quantify and hence clarify their objectives, says John Hough, principal technical advisor on biodiversity for the United Nations Development Programme. ‘We know what we don't want,’ he says, ‘but we're not very good at saying what we do want.’ A hypothetical model simulating how Madagascar should distribute an annual conservation budget of $4 million reveals that direct payments would have protected some 80% of original forest compared with only 12% protected through a system of indirect incentives. What's more, the annual income of rural residents would have been twice that generated through indirect investment (Conrad and Ferraro 2001). For Ferraro, the logic of direct payment is simple. He draws an analogy with a car journey from A to B. There are two routes that will bring you to B, one circuitous and the other direct. If you only have a single tank of fuel, opting for the direct route improves the likelihood you will arrive at your destination. An indirect approach to conservation is like taking the circuitous route, he says, and the chances are that you will run out of fuel. But if it's that simple, why are governments, non-governmental organisations, private bodies, and international organisations not jumping at the chance to experiment with this approach? Paying in Perpetuity There are those that have reservations about direct payments. The distinction between indirect and direct interventions is artificial, says Thomas Lovejoy, president of the Heinz Center, a nonprofit institution dedicated to improving the scientific and economic foundation for environmental policy. ‘In some cases, direct payment is the only way conservation can happen,’ he says. ‘In others, the indirect is important to reinforce a situation where there already is conservation. In yet others both are needed.’ Sjaak Swart of the Section of Science and Society at Groningen University in The Netherlands argues that if conservation is to succeed, it must be rooted in the hearts and minds of those involved. Direct payments create a vision of nature dominated by calculable, monetary concerns, he says. This approach can only work in the short term, he argues, and indirect tools like debate and education are needed to involve communities in the long term. ‘You need the commitment of the local people to save the biodiversity of our world,’ he says. Marine biologist Steve Trott agrees. He is project coordinator for the Local Ocean Trust, a charity-based conservation organisation operating in the Watamu and Malindi Marine Parks and Reserve in Kenya (http://www.watamuturtles.com), and is using direct payments to help reduce the slaughter of turtles by local fishermen. The Watamu Turtle Watch Program is currently paying fishermen just over $3 a turtle to release the animals from their nets rather than kill them. Before the scheme started in 2000, only around 50 turtles were being released from nets each year. By 2003, more than 500 a year were making it back into the sea. Elsewhere along the Kenyan coast, where fishermen do not get these payments, turtles continue to be killed, says Trott. However, the financial incentives are only part of a grander program of education and support to sensitise people to the conservation message, he says. Eventually, the plan is to stop payments altogether. ‘Payment will be reduced as education and awareness is increased to the point where it's phased out,’ he says. Reducing or stopping the payment could work, says Ferraro, but it is more likely that the turtles will begin to suffer once more. ‘If I had to wager, I'd bet people would go back to their old patterns eventually.’ This means that direct payments require an ongoing financial commitment, and many people don't like this idea, he says. To the Test The idea of direct payments needs empirical testing before it can be embraced with confidence, admits Ferraro. Funding bodies should demand experimental and control data to allow the success of an intervention to be gauged. Conservation biologists must therefore be trained in the skills needed to collect and evaluate these data. ‘Without adequate data and controls you're only going to be left with guesses and vague anecdotes about the effects of a program intervention,’ he says. Decision makers should begin to design controlled experiments from which they can make inferences about the effectiveness of these different interventions, he suggests. There are other drawbacks of direct payments. One concern is that they might just shift the pressure from one site to another that was not previously being exploited. Furthermore, in developing countries, land tenure is often ambiguous, which can make investment an unattractive prospect for funding agencies—they want to be sure they know where their money is going. But, notes Ferraro, such objections also apply to indirect interventions. ‘I don't necessarily believe that conservation payments will be successful,’ he says. ‘It's more I believe that of all the ideas out there for protecting biodiversity, this is the least bad.’ All this talk of cost, benefit, and efficiency is creeping into conservation speak. For some, these cold and calculating terms are an odd way to describe the world's wonderfully unpredictable wildlife. But, increasingly, there are calls for conservation biology to cast aside its sentimental demons: biodiversity is a commodity that can be bought and sold; conservation is business. Box 1. The Cucumber Conflict At the beginning of the 1990s, fishermen in the Galápagos began collecting sea cucumbers from the waters around their islands to meet ongoing demand for these aphrodisiac ‘earthworms of the sea’ in Southeast Asia (Figure 3). Others intent on taking advantage of this commercial opportunity began to arrive from the Ecuadorian mainland in their hundreds. In 1998, Ecuador's president signed the Special Law of the Galápagos, which created the Galápagos Marine Reserve, protecting its waters from commercial fishing and imposed restrictions on domestic immigration. But by then, too many were already intent on reaping the financial rewards the sea cucumber promised them—by the end of the decade, a single sea cucumber could fetch nearly $2. Conservation biologists at the Charles Darwin Research Station on the central island of Santa Cruz worked out levels of fishing that might be sustainable. In 1999—the first season in which sea cucumber fisheries were monitored and regulated—nearly 800 fishermen collected more than 4 million animals worth more than $3.4 million in a short two-month window. In January 2000, fishermen protesting the closure of the fishery took over offices of the Galápagos National Park Service and Charles Darwin Research Station, holding humans and animals hostage. Figure 3 The Prized Galápagos Sea Cucumber, Stichopus fuscus Courtesy of Henry Nicholls. Box 2. Paying for Forests The longest-running and best-known example of a direct-payment initiative is the Programa de Pago de Servicios Ambientales (PSA) in Costa Rica (Figure 4). In the second half of the 20th century, forest cover in Costa Rica fell from around 50% to 25%, and more than half of what remained was on unprotected, privately owned land. In 1996, the PSA was set up to make direct payments to individual landowners, associations of landowners, or indigenous reserves in exchange for ‘environmental services’—anything from forest conservation to providing a supply of water. Some 85% of contracts have been for forest conservation. By 2001, more than 2,800 km2 were protected by payments of $4,000 per km2 every year, and contracts covering a further 8,000 km2 were being processed. Most of the money for these payments comes from a petrol tax on Costa Rican citizens, although the Global Environmental Facility has put up money for biodiversity conservation, Costa Rica's Office of Joint Implementation has paid for carbon sequestration, and domestic hydroelectricity and municipal water providers pay for water services. Figure 4 Forest Protected by Costa Rica's PSA Courtesy of Subhrendu Pattanayak. Henry Nicholls is a freelance science writer based in London, United Kingdom. E-mail: [email protected] Abbreviation PSAPrograma de Pago de Servicios Ambientales ==== Refs References and Further Reading Balmford A Whitten T Who should pay for tropical conservation and how the costs should be met Oryx 2004 37 238 250 Balmford A Gaston KJ Blyth S James A Kapos V Global variation in terrestrial conservation costs, conservation benefits, and unmet conservation needs Proc Natl Acad Sci U S A 2003 100 1046 1050 12552123 Conrad JC Ferraro PJ Habitat conservation: The dynamics of direct and indirect payments Environmental Policy Working Paper Ser 2001 Georgia State University 2001–005. Available: http://epp.gsu.edu/pferraro/docs/ConradFerraroWorkingPaper2001Distrib.pdf via the Internet. Accessed 21 July 2004 du Toit JT Walker BH Campbell BM Conserving tropical nature: Current challenges for ecologists Trends Ecol Evol 2004 19 12 17 16701220 Ferraro PJ Kiss A Direct payments to conserve biodiversity Science 2002 298 1718 1719 12459569 Ferraro PJ Simpson RD Protecting forests and biodiversity: Are investments in eco-friendly production activities the best way to protect endangered species and enhance rural livelihoods? Paper presented at the International Conference on Rural Livelihoods, Forests and Biodiversity 2004 Bonn, Germany 19 23 May 2003. Available: http://www.cifor.cgiar.org/publications/corporate/cd-roms/bonn-proc/pdfs/papers/t4_final_ferraro.pdf via the Internet. Accessed 21 July 2004 Kiss A Is community-based ecotourism a good use of biodiversity conservation funds? Trends Ecol Evol 2004 19 232 237 16701261 Swart JAA Will direct payments help biodiversity? Science 2003 299 1981 1982 12663896

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PLoS Biol. 2004 Sep 14; 2(9):e310

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PLoS Biol

10.1371/journal.pbio.0020310

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==== Front PLoS BiolPLoS BiolpbioplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 10.1371/journal.pbio.0020311PrimerBotanyPlant SciencePlantsArabidopsisHormonal Regulation of Plant Growth and Development PrimerGray William M 9 2004 14 9 2004 14 9 2004 2 9 e311Copyright: © 2004 William M. Gray.2004This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. Interdependency of Brassinosteroid and Auxin Signaling in Arabidopsis Besides environmental factors, plant growth depends upon endogenous signals. Bill Gray examines what these hormonal signals are and how they act to regulate many aspects of growth and development. ==== Body Phytohormones: What Are they? Plant growth and development involves the integration of many environmental and endogenous signals that, together with the intrinsic genetic program, determine plant form. Fundamental to this process are several growth regulators collectively called the plant hormones or phytohormones. This group includes auxin, cytokinin, the gibberellins (GAs), abscisic acid (ABA), ethylene, the brassinosteroids (BRs), and jasmonic acid (JA), each of which acts at low concentrations to regulate many aspects of plant growth and development. With the notable exception of the steroidal hormones of the BR group, plant hormones bear little resemblance to their animal counterparts (Figure 1). Rather, they are relatively simple, small molecules such as ethylene gas and indole-3-acetic acid (IAA), the primary auxin in the majority of plant species. The concept of plant hormones originates from a classical experiment on phototropism, the bending of plants toward light, carried out by Charles Darwin and his son Francis in 1880. The Darwins were able to demonstrate that when oat seedlings were exposed to a lateral light source, a transported signal originating from the plant apex promoted differential cell elongation in the lower parts of the seedling that resulted in it bending toward the light source. This signal was subsequently shown to be IAA, the first known plant hormone. Figure 1 Chemical Structures of the Plant Hormones A partial list of the responses elicited by each hormone is provided below. Ethylene gas promotes fruit ripening, senescence, and responses to pathogens and abiotic stresses. IAA (an auxin) regulates cell division and expansion, vascular differentiation, lateral root development, and apical dominance. Cytokinins are adenine derivatives first identified by their ability to promote cytokinesis. JA is a volatile signal that modulates pollen development and responses to pathogen infection. The BRs regulate cell expansion and photomorphogenesis (light-regulated development). GAs are diterpenoid compounds that promote germination, stem elongation, and the induction of flowering. ABA promotes seed dormancy and is involved in several stress signaling pathways. What Do They Do? Virtually every aspect of plant growth and development is under hormonal control to some degree. A single hormone can regulate an amazingly diverse array of cellular and developmental processes, while at the same time multiple hormones often influence a single process. Well-studied examples include the promotion of fruit ripening by ethylene, regulation of the cell cycle by auxin and cytokinin, induction of seed germination and stem elongation by GA, and the maintenance of seed dormancy by ABA. Historically, the effects of each hormone have been defined largely by the application of exogenous hormone. More recently, the isolation of hormone biosynthetic and response mutants has provided powerful new tools for painting a clearer picture of the roles of the various phytohormones in plant growth and development. How Do They Work? Plant biologists have been fascinated by the regulatory capacity of phytohormones since the time of their discovery, and the notion that hormone levels or responses could be manipulated to improve desired plant traits has long been an area of intense interest. Perhaps the best-known example of this is the isolation of dwarf varieties of wheat and rice that led to the “green revolution” in the second half of the 20th century, which is credited with saving millions of people around the globe from starvation. These dwarf varieties have shorter stems than wild-type, making these plants less susceptible to damage by wind and rain. The molecular isolation of these “dwarfing genes” has revealed that they encode components of the GA biosynthesis and response pathways (Peng et al. 1999; Sasaki et al. 2002). To elucidate the molecular mechanisms underlying phytohormone action, several researchers have utilized the genetically facile model plant Arabidopsis thaliana to isolate mutations that confer altered response to applied hormone. Molecular and biochemical analysis of the gene products defined by these mutations, coupled with expression studies aimed at identifying the downstream target genes that mediate hormonal changes in growth and development, has begun to unlock some of the mysteries behind phytohormone action. While no hormone transduction pathway is completely understood, we now have a rudimentary understanding of many of the molecular events underlying hormone action. Several reviews covering the individual hormone pathways in greater detail have recently been published (Turner et al. 2002; Gomi and Matsuoka 2003; Himmelbach et al. 2003; Kakimoto 2003; Dharmasiri and Estelle 2004; Guo and Ecker 2004; Wang and He 2004). Common Themes Regulation by proteolysis has emerged as a resounding theme in plant hormone signaling. The ubiquitin-mediated degradation of key regulatory proteins has been demonstrated, or is at least likely, for all of the phytohormone response pathways (Smalle and Vierstra 2004). In the case of auxin, the response pathway is normally subject to repression by a large family of transcriptional regulators called the Aux/IAA proteins (Figure 2). These proteins dimerize with members of the auxin response factor (ARF) family of transcription factors, thus preventing ARFs from activating auxin-responsive genes (Tiwari et al. 2004). Upon an auxin stimulus, an SCF (SKP1/Cullin/F-box protein) ubiquitin ligase (Deshaies 1999) containing the TIR1 F-box protein ubiquitinates the Aux/IAA proteins, marking them for degradation by the 26S proteasome thereby de-repressing the response pathway (Gray et al. 2001). The hormone promotes the Aux/IAA–TIR1 interaction; however, the molecular mechanisms behind this regulation are unclear. Most yeast and animal SCF substrates must be post-translationally modified, usually by phosphorylation, before they are recognized by their cognate F-box protein. Despite numerous efforts to identify auxin-induced modification of Aux/IAA proteins, no such signal has been discovered, raising the distinct possibility that auxin uses a novel mechanism to regulate SCF–substrate interactions. Figure 2 The Ubiquitin-Mediated Proteolysis of Aux/IAA Proteins Regulates Auxin Response (A) Wild-type Arabidopsis thaliana and the axr2-1 mutant. axr2-1 is a dominant gain-of-function mutation in an Aux/IAA gene that confers reduced auxin response. The mutant axr2-1 protein constitutively represses auxin response because it cannot be targeted for proteolysis by the SCFTIR1 ubiquitin ligase. The effect of the mutation on AXR2 stability is shown in a pulse-chase experiment (inset). Wild-type and axr2-1 seedlings were labeled with 35S-methionine and AXR2/axr2-1 protein was immunoprecipitated either immediately after the labeling period (t = 0) or following a 15-minute chase with unlabeled methionine (t = 15). (B) A simplified model for auxin response. In the absence of an auxin stimulus, Aux/ IAA proteins inhibit ARF transcriptional activity by forming heterodimers. Auxin perception (by an unknown receptor) targets the Aux/IAA proteins to the SCFTIR1 complex, resulting in their ubiquitination and degradation, thereby de-repressing the ARF transcription factors. Among the ARF targets are the Aux/IAA genes themselves, which produce nascent Aux/IAA proteins that restore repression upon the pathway in a negative feedback loop. Ethylene and cytokinin are both perceived by receptors sharing similarity to bacterial two-component regulators. Common in prokaryotes, but apparently restricted to plants and fungi in eukaryotes, these modular signaling systems involve a membrane-bound receptor containing an intracellular histidine kinase (HK) domain (Wolanin et al. 2002). Ligand binding activates the kinase, resulting in autophosphorylation and initiation of a series of phosphotransfer reactions that culminates with the activation of a response regulator protein that functions as the effector component of the pathway. Cytokinin signaling appears to largely follow this paradigm (Kakimoto 2003). Ethylene response, however, appears more complex (Guo and Ecker 2004). Ethylene is perceived by a family of five receptors. ETR1 and ERS1 contain a consensus HK domain, however, the HK domains of ETR2, ERS2, and EIN4 are degenerate and lack elements necessary for catalytic activity. This fact, together with studies of “kinase-dead” mutants of ETR1, suggests that HK activity is not required for ethylene response. Mutations that abolish ethylene binding in any of the five receptor genes are dominant and confer ethylene insensitivity, indicating that the receptors function as negative regulators of the ethylene pathway. Genetic and molecular studies have positioned these receptors upstream of the Raf-like MAP kinase kinase kinase, CTR1, which interacts with the receptors and also acts as a negative regulator (Figure 3). The integral membrane protein, EIN2, and the transcription factors EIN3 and EIL1 are positive regulators of ethylene signaling downstream of CTR1. Current models propose that hormone binding inactivates the receptors, thus resulting in down-regulation of CTR1 activity. Since the identification of CTR1, biologists have speculated that a MAP kinase cascade may be involved. Only recently, however, have putative MAP kinase kinase and MAP kinase components of the ethylene pathway been identified (Chang 2003). Interestingly, these kinases appear to positively regulate ethylene response, suggesting that CTR1 must inhibit their function. If so, this would represent a novel twist on the traditional MAP kinase signaling paradigm. Precisely how the ethylene signal is transduced to the EIN3 and EIL1 transcription factors remains unclear. However, the recent finding that ethylene stabilizes these transcription factors, which are targeted for degradation by an SCF complex in the absence of ethylene, clearly indicates a role for the ubiquitin pathway (Guo and Ecker 2003; Potuschak et al. 2003). One of the known targets for EIN3 is the ERF1 transcription factor, which activates several genes involved in a subset of ethylene responses. Figure 3 A Model for the Arabidopsis Ethylene Response Pathway Ethylene is perceived by a family of two-component receptors containing a consensus (unshaded) or degenerate (shaded) HK domain (H). Three of the receptors also contain a C-terminal receiver domain (R). The receptors negatively regulate ethylene response together with CTR1 in a complex on the endoplasmic reticulum membrane. Perception results in reduced receptor and CTR1 activities and activation of a MAP kinase kinase, which transmits the signal through the EIN2 membrane protein, ultimately resulting in the activation of a transcriptional cascade in the nucleus. The EIN3 and EIL1 transcription factors regulate primary response genes including ERF1, which activates a subset of secondary ethylene-induced genes involved in defense responses. EIN3/EIL1 abundance is regulated in an ethylene-dependent manner by SCF complexes containing F-box proteins encoded by the ethylene-induced genes EBF1 and EBF2. Positive- and negative-acting components of the pathway are indicated in green and red, respectively. Solid lines indicate regulation that is likely to be through direct interactions. Dotted lines indicate speculative interactions based on genetic studies. Signal Integration and Combinatorial Control Long ago, plant physiologists noted the apparent antagonistic interactions between some of the phytohormones, such as between auxin and cytokinin in the regulation of root–shoot differentiation and between GA and ABA in germination. Other processes are synergistically regulated by multiple hormones. While it has long been obvious that hormones do not function in discrete pathways, but rather exhibit extensive cross-talk and signal integration with each other and with environmental and developmental signaling pathways, the molecular basis for such coordinated regulation has been unclear. Several recent findings have begun to elucidate the molecular details of some of these events. One example of such signal integration was recently described for the ethylene and JA pathways (Lorenzo et al. 2003). Genetic studies had previously implicated both hormones as important regulators of pathogen defense responses, as well as of the wounding response and other stress-related pathways. Additionally, microarray analysis has identified a large number of genes that are responsive to both hormones. The ERF1 transcription factor was recently found to be an intersection point for these two signaling pathways (Lorenzo et al. 2003). Like ethylene, JA rapidly induces ERF1 expression, and treatment with both hormones synergistically activates ERF1. Induction of ERF1 by both hormones alone or in combination is dependent upon both signaling pathways, and constitutive overexpression of ERF1 rescues the defense-response defects of both ethylene- and JA-insensitive mutants. These findings suggest that ERF1 represents one of the first signaling nodes identified in the complex web of hormonal cross-talk. The auxin and BR pathways also appear to converge and mutually regulate some developmental processes. Both hormones promote cell expansion, and microarray studies have revealed that as many as 40% of all BR-induced genes are also up-regulated by auxin (Goda et al. 2004; Nemhauser et al. 2004). BR is perceived by the cell surface receptor kinase BRI1 (Wang and He 2004). The SHAGGY/GSK3-type kinase BIN2 acts as a negative regulator of the pathway downstream of the receptor. In the absence of a BR signal, BIN2 phosphorylates the transcription factors BES1 and BZR1, targeting them for proteolysis by the 26S proteasome. Upon a BR stimulus, BIN2 is inactivated, allowing BES1 and BZR1to accumulate in the nucleus, where they are presumably involved in regulating BR-responsive genes. Using combined genetic, physiological, and genomic approaches, Nemhauser and colleagues (2004) were able to demonstrate that auxin and BR regulate Arabidopsis hypocotyl (embryonic stem) elongation in a synergistic and interdependent fashion. Elevating endogenous auxin levels rendered plants more sensitive to BR application in hypocotyl elongation assays, and this response was dependent upon both the auxin and BR signaling pathways. Genetic studies suggest that the convergence of these two pathways occurs at a late point in hormone signaling, perhaps at the promoters of the many genes responsive to both hormones. In support of this notion, bioinformatic analysis identified distinct sequence elements that were enriched specifically in the promoters of auxin-induced, BR-induced, and auxin/BR-induced genes. Many Unanswered Questions While great strides have been made in recent years in understanding the molecular basis of phytohormone action, many fundamental questions remain. Receptors and other upstream signaling components remain to be identified for the majority of the phytohormones. Equally important are the elucidation of hormonal networks and the integration of these networks with the morphogenetic program, such that our understanding of hormone action can be placed in a developmental context. The author wishes to thank members of his lab for helpful comments on this manuscript. Work in the author's laboratory on auxin response is supported by National Institutes of Health grant GM067203 and the Mcknight Foundation. William M. Gray is in Plant Biology at the University of Minnesota, St. Paul, Minnesota, United States of America. E-mail: [email protected] Abbreviations ABAabscisic acid ARFauxin response factor BRbrassinosteroid GAgibberellin HKhistidine kinase IAAindole-3-acetic acid JAjasmonic acid SCFSKP1/Cullin/F-box protein ==== Refs References Chang C Ethylene signaling: The MAPK module has finally landed Trends Plant Sci 2003 8 365 368 12927968 Deshaies RJ SCF and Cullin/Ring H2-based ubiquitin ligases Annu Rev Cell Dev Biol 1999 15 435 467 10611969 Dharmasiri N Estelle M Auxin signaling and regulated protein degradation Trends Plant Sci 2004 9 302 308 15165562 Goda H Sawa S Asami T Fujioka S Shimada Y Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis Plant Physiol 2004 134 1555 1573 15047898 Gomi K Matsuoka M Gibberellin signalling pathway Curr Opin Plant Biol 2003 6 489 493 12972050 Gray WM Kepinski S Rouse D Leyser O Estelle M Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins Nature 2001 414 271 276 11713520 Guo H Ecker JR Plant responses to ethylene gas are mediated by SCF(EBF1/EBF2)-dependent proteolysis of EIN3 transcription factor Cell 2003 115 667 677 14675532 Guo H Ecker JR The ethylene signaling pathway: New insights Curr Opin Plant Biol 2004 7 40 49 14732440 Himmelbach A Yang Y Grill E Relay and control of abscisic acid signaling Curr Opin Plant Biol 2003 6 470 479 12972048 Kakimoto T Perception and signal transduction of cytokinins Annu Rev Plant Biol 2003 54 605 627 14503005 Lorenzo O Piqueras R Sanchez-Serrano JJ Solano R ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense Plant Cell 2003 15 165 178 12509529 Nemhauser JL Mockler TC Chory J Interdependency of brassinosteroid and auxin signaling in Arabidopsis PLoS Biol 2004 2 e258 15328536 Peng J Richards DE Hartley NM Murphy GP Devos KM ‘Green revolution’ genes encode mutant gibberellin response modulators Nature 1999 400 256 261 10421366 Potuschak T Lechner E Parmentier Y Yanagisawa S Grava S EIN3-dependent regulation of plant ethylene hormone signaling by two arabidopsis F box proteins: EBF1 and EBF2 Cell 2003 115 679 689 14675533 Sasaki A Ashikari M Ueguchi-Tanaka M Itoh H Nishimura A Green revolution: A mutant gibberellin-synthesis gene in rice Nature 2002 416 701 702 11961544 Smalle J Vierstra RD The ubiquitin 26s proteasome proteolytic pathway Annu Rev Plant Physiol Plant Mol Biol 2004 55 555 590 Tiwari SB Hagen G Guilfoyle TJ Aux/ IAA proteins contain a potent transcriptional repression domain Plant Cell 2004 16 533 543 14742873 Turner JG Ellis C Devoto A The jasmonate signal pathway Plant Cell 2002 14 S153 S164 12045275 Wang ZY He JX Brassinosteroid signal transduction—Choices of signals and receptors Trends Plant Sci 2004 9 91 96 15102375 Wolanin PM Thomason PA Stock JB Histidine protein kinases: Key signal transducers outside the animal kingdom Genome Biol 2002 3 REVIEWS3013 12372152

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1181533934610.1186/1471-2105-5-118Methodology ArticleEstimating mutual information using B-spline functions – an improved similarity measure for analysing gene expression data Daub Carsten O [email protected] Ralf [email protected] Joachim [email protected] Sebastian [email protected] Max Planck Institute of Molecular Plant Physiology, Potsdam, 14424, Germany2 Nonlinear Dynamics Group, Institute of Physics, University of Potsdam, Potsdam, 14415, Germany3 Scienion AG, Volmerstrasse 7a, Berlin, 12489, Germany4 Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, 17177, Sweden2004 31 8 2004 5 118 118 15 12 2003 31 8 2004 Copyright © 2004 Daub et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The information theoretic concept of mutual information provides a general framework to evaluate dependencies between variables. In the context of the clustering of genes with similar patterns of expression it has been suggested as a general quantity of similarity to extend commonly used linear measures. Since mutual information is defined in terms of discrete variables, its application to continuous data requires the use of binning procedures, which can lead to significant numerical errors for datasets of small or moderate size. Results In this work, we propose a method for the numerical estimation of mutual information from continuous data. We investigate the characteristic properties arising from the application of our algorithm and show that our approach outperforms commonly used algorithms: The significance, as a measure of the power of distinction from random correlation, is significantly increased. This concept is subsequently illustrated on two large-scale gene expression datasets and the results are compared to those obtained using other similarity measures. A C++ source code of our algorithm is available for non-commercial use from [email protected] upon request. Conclusion The utilisation of mutual information as similarity measure enables the detection of non-linear correlations in gene expression datasets. Frequently applied linear correlation measures, which are often used on an ad-hoc basis without further justification, are thereby extended. ==== Body Background The evaluation of the complex regulatory networks underlying molecular processes poses a major challenge to current research. With modern experimental methods in the field of gene expression, it is possible to monitor mRNA abundance for whole genomes [1,2]. To elucidate the functional relationships inherent in this data, a commonly used approach is the clustering of co-expressed genes [3]. In this context, the choice of the similarity measure used for clustering, as well as the clustering method itself, is crucial for the results obtained. Often, linear similarity measures such as the Euclidean distance or Pearson correlation are used in an ad-hoc manner. By doing so, it is possible that subsets of non-linear correlations contained in a given dataset are missed. Therefore, information theoretic concepts, such as mutual information, are being used to extend more conventional methods in various contexts ranging from expression [4-8] and DNA sequence analysis [9,10], to reverse engineering [11] and independent component analysis [12,13]. Also aside the bioinformatics field, mutual information is widely utilised in diverse disciplines, such as physics [14], image recognition [15], speech recognition [16], and various others. In extension to other similarity measures, mutual information provides a general measure of statistical dependence between variables. It is thereby able to detect any type of functional relationship, extending the potentialities of linear measures as illustrated in Figure 1. In this work, we discuss mutual information as a measure of similarity between variables. In the first section, we give a short introduction into the basic concepts including a brief description of the commonly used approaches for numerical estimation from continuous data. In the following section, we then present an algorithm for estimating mutual information from finite data. The properties arising from this approach are compared to previously existing algorithms. In subsequent sections, we then apply our concept to large-scale cDNA abundance datasets and determine if these datasets can be sufficiently described using linear measurements or if a significant amount of non-linear correlations are missed. Mutual information Mutual information represents a general information theoretic approach to determine the statistical dependence between variables. The concept was initially developed for discrete data. For a system, A, with a finite set of M possible states {a1, a2, ... , }, the Shannon entropy H(A) is defined as [17] where p(ai) denotes the probability of the state ai. The Shannon entropy is a measure for how evenly the states of A are distributed. The entropy of system A becomes zero if the outcome of a measurement of A is completely determined to be aj, thus if p(aj) = 1 and p(ai) = 0 for all i ≠ j, whereas the entropy becomes maximal if all probabilities are equal. The joint entropy H(A, B) of two systems A and B is defined analogously This leads to the relation H(A, B) ≤ H(A) + H(B)     (3) which fulfils equality only in the case of statistical independence of A and B. Mutual information MI(A, B) can be defined as [17] MI(A, B) = H(A) + H(B) - H(A, B) ≥ 0     (4) It is zero if A and B are statistically independent and increases the less statistically independent A and B are. If mutual information is indeed to be used for the analysis of gene-expression data, the continuous experimental data need to be partitioned into discrete intervals, or bins. In the following section, we briefly review the established procedures; a description of how we have extended the basic approach will be provided in the subsequent section. Estimates from continuous data In the case of discrete data the estimation of the probabilities p(ai) is straightforward. Many practical applications, however, supply continuous data for which the probability distributions are unknown and have to be estimated. In a widely used approach [7], the calculation of mutual information is based on the binning of data into M discrete intervals ai, i = 1... MA. For experimental data consisting of N measurements of a variable xu, u = 1... N, an indicator function Θi counts the number of data points within each bin. The probabilities are then estimated based on the relative frequencies of occurrence with For two variables the joint probabilities are calculated analogously from a multivariate histogram. Additionally it has been suggested [14] to adaptively choose the sizes of the bins, so that each bin constructed nearly has a uniform distribution of points. In a different approach, kernel methods are used for the estimation of the probability density of Eq. (5) [18-20]. Entropies are then calculated by integration of the estimated densities. Recently, an entropy estimator was suggested [21] and showed in an extensive comparison to other commonly used estimators to be superior. Results Fuzzy mutual information In the classical binning approach, described above, each data point is assigned to one, and only one, bin. For data points near to the border of a bin, small fluctuations due to biological or measurement noise might shift these points to neighbouring bins. Especially for datasets of moderate size, the positions of the borders of the bins can thereby strongly affect the resulting mutual information [18]. In a manner analogous to kernel density estimators (KDE), we now present a generalisation to the classical binning in which we aim to overcome some of the drawbacks associated with the simple approach. Within our algorithm, we allow the data points to be assigned to several bins simultaneously. For this, we extended the indicator function Θ(x) to the set of polynomial B-spline functions. Here, we do not provide the mathematical details for these functions since they have been discussed extensively in the literature [22-24], but rather focus on the practical applicability. Within the B-spline approach, each measurement is assigned to more than one bin, i, with weights given by the B-spline functions Bi,k. The spline order k determines the shape of the weight functions and thereby the number of bins each of the data points is assigned to. A spline order k = 1 corresponds to the simple binning, as described in the previous section: Each data point is assigned to exactly one bin (Figure 2, left). For k = 3, each data point is assigned to three bins, with the respective weights given by the values of the B-spline functions at the data point (Figure 2, right). B-spline functions The first step in the definition of the B-spline functions is the definition of a knot vector ti for a number of bins i = 1... M and one given spline order k = 1... M - 1 [22] where the spline order determines the degree of the polynomial functions. The domain of the B-spline functions lies in the interval z ∈ [0, M - k + 1]. To cover the range of the variables, the new indicator function based on the B-spline functions needs to be linearly transformed to map their range. The recursive definition of the B-spline functions are as follows [22] An important property of B-spline functions is the implicit standardisation of coefficients: All weights belonging to one data point sum up to unity. Algorithm Input • Variables x and y with values xu and yu, u = 1... N • Bins ai, i = 1... Mx and bj, j = 1... My • Spline order k Output • Mutual information between variable x and y Algorithm 1. Calculation of marginal entropy for variable x (a) Determine with (b) Determine Mx weighting coefficients for each xu from (c) Sum over all xu and determine p(ai) for each bin ai from (d) Determine entropy H(x) according to Eq. (1) 2. Calculation of joint entropy of two variables x and y (a) Apply steps 1 (a) and (b) to both variables x and y, independently (b) Calculate joint probabilities p(ai, bj) for all Mx × My bins according to (c) Calculate the joint entropy H(x,y) according to Eq. (2) 3. Calculate the mutual information MI(x,y) according to Eq. (4) Example We show the estimation with the standard binning and our approach ex-emplarily on two artificial variables x = 0.0,0.2,0.4,0.6,0.8,1.0 and y = 0.8,1.0,0.6,0.4,0.0,0.2 for M = 3 bins, spline order k = 2, and the logarithm to basis two. Simple binning For both variables, each of the three histogram bins contains two values p(a1) = p(a2) = p(a3) = , analogously for p(bi) due to the symmetry of data H(x) = H(y) = = log2 3 ≈ 1.58. For the calculation of the joint probability, three of the nine two dimensional bins contain two values each p(a1, b3) = p(a2, b2) = p(a3, b1) = resulting in H(x, y) = log2 3 and MI(x, y) = log2 3. B-spline approach The modified indicator function is determined to Bi,k(2x) according to Eq. (9) (rule 1(a)). For each value xu three weighting coefficients are determined (rule 1(c)) and probabilities are calculated (rule 1(d)) (Table 1). The analogous procedure is applied to variable y and the single entropies are calculated to H(x) = H(y) = Iog2(10) - 0.61og2(3) - 0.41og2(4) ≈ 1.57. Both, H(A) and H(B), are slightly smaller than the entropies calculated from the simple binning. The joint probabilities are p(a1, b1) = p(a3, b3) = 0, p(a1, b2) = p(a2, b1) = p(a2, b3) = p(a3, b2) = 0.56/6, p(a1, b3) = p(a3, b1) = 1.24/6, p(a2, b2) = 1.28/6 (rule 2 (b)) resulting in H(x,y) = 2.7 and MI(x,y) = 0.45. In the next sections, we discuss some of the properties arising from the utilisation of B-spline functions for the estimation of mutual information and compare our approach to other commonly used estimators. We support this discussion using examples for which the underlying distributions and thereby the true mutual information is known. Size of data It has been discussed elsewhere [25-28,20] that the estimated mutual information is systematically overestimated for a finite size of N data points. For the simple binning approach, the mean observed mutual information can be calculated explicitly as the deviation from the true mutual information As can be seen for an example of artificially generated equidistributed random numbers (Figure 3, left), mutual information calculated from the simple binning scales linearly with 1/N, with the slope depending on the number of bins M in accordance with Eq. (12). Figure 3 shows that this scaling is preserved for the extension to B-spline functions, while the slope is significantly decreased for k = 3, compared to the estimation with the simple binning (k = 1). Mutual information calculated from KDE does not show a linear behaviour but rather an asymptotic one with a linear tail for large datasets. The values are slightly increased compared to the ones from the B-spline approach. The entropy estimator gives values comparable to the ones obtained from the B-spline approach. More importantly, a similar result also holds for the standard deviation of mutual information. As shown in Figure 3 (right), the standard deviation of the mutual information estimated with the simple binning (k = 1) scales with 1/N for statistically independent events [26,29]. For the B-spline approach (k = 3), this scaling still holds, but the average values are decreased significantly. For the KDE approach, an asymptotic run above the values from the B-spline approach is observed, again with linear tail for large datasets. shows a linear scaling slightly below the simple binning. The spline order The interpretation of any results obtained from the application of mutual information to experimental data is based on testing to see if the calculated results are consistent with a previously chosen null hypothesis. By following the intuitive approach that the null hypothesis assumes the statistical independence of variables, mutual information is tested against a surrogate dataset, which is consistent with this null hypothesis. As discussed previously in more detail [20], one way of generating such a surrogate dataset is by random permutations of the original data. From the mutual information of the original dataset MI(X,Y)data, the average value obtained from surrogate data <MI(Xsurr, Ysurr) >, and its standard deviation σsurr, the significance S can be formulated as For each S the null hypothesis can be rejected to a certain level α depending on the underlying distribution. With increasing significance the probability of false positive associations drops. In the following, we address the influence of the spline order and the number of bins on the estimation of mutual information. Based on 300 data points of an artificially-generated dataset drawn from the distribution shown in Figure 1, we calculate the mutual information for M = 6 bins and different spline orders k = 1... 5 (Figure 4, left). From 300 shuffled realisations of this dataset, the mean and maximum mutual information are shown with the standard deviation as error-bars. For all spline orders the null hypothesis can be rejected, in accordance with the dataset shown in Figure 1. To estimate the strength of the rejection, we calculate the significance according to Eq. (13) (Figure 4, right). It can be observed that the largest change in the significance of the mutual information occurs in the transition from k = 1 (simple boxes) to k = 2 with an increase by roughly two-fold. Using more sophisticated functions (k ≥ 3) does not further improve the significance. Similar findings have been reported in the context of kernel density estimators [19]. The major contribution leading to this increase of the significance is given by the distribution of surrogate data which becomes more narrow for k > 1 leading to smaller standard deviations σsurr. The same dataset is used to show the dependency of mutual information on the number of bins for two spline orders k = 1 and k = 3 (Figure 5). Mutual information estimated from data as well as from surrogate data shows a robust run without strong fluctuations within the range of bins shown. From this we can conclude that the choice of the number of bins does not affect the resulting mutual information notably as long as it is chosen to be within a reasonable range. Again, the significance is calculated (Figure 6) and compared to the significances obtained from the KDE approach and the estimator. It can be observed that the significance of the mutual information calculated with B-spline functions increased roughly by two-fold compared to the simple binning. The significance obtained from KDE is not depending on M and was determined to be similar to the significance estimated from the B-spline approach. The numerically expensive integration of KDE, however, limits the size of utilisable datasets. The KDE run time requirements were (104) times higher than the ones from the B-spline approach. Strategies to simplify the integration step were proposed [20] but have to be used with caution since they assume particular properties of the distribution of experimental data that are in general not fulfilled. The recently introduced entropy estimator produces intermediate significances between the ones from the binning and the B-spline approach for higher bin numbers. For low bin numbers, the significances are relatively poor. Application on data We now turn to the analysis of experimentally measured gene expression data. As shown previously, the application of mutual information to large-scale expression data reveals biologically-relevant clusters of genes [7,30]. In this section, we will not repeat these analyses, but determine if the correlations detected using mutual information are missed using the established linear measures. Among the most frequently used measures of similarity for clustering co-expressed genes are the Euclidean distance and the Pearson correlation coefficient R [3]. If correlations are well described by the Pearson correlation and the distribution of data is approximately Gaussian like, the relationship between the mutual information and the Pearson correlation given by [32] is expected to be fulfilled. Therefore, we calculated both, the mutual information and the Pearson correlation, for two large-scale gene expression datasets (Figure 7). For each pair of genes X and Y we plot the tuple (MI(X,Y), R(X,Y)). In order to address significance, we additionally calculate all tuples from shuffled data. The first dataset contains cDNA measurements for S. cerevisiae for up to E1 = 300 experiments [31]. To avoid numerical effects arising from different numbers of defined expression values (missing data points) for each gene, we exclusively utilised genes that are fully defined for all experimental conditions resulting in G1 = 5345 genes. Analysis on this dataset using mutual information has been done before [20,32] on rank-ordered data. The rank-ordering lead to homogeneously distributed data and thereby enabled the application of a simplified algorithm for the numerical estimation from kernel density estimators. The utilisation of our B-spline approach allows us to extend this analysis to non rank-ordered data thereby keeping the original distribution of experimental data. In contrast to the previous studies we find for non rank-ordered data that the theoretical prediction of Eq. 14 is no longer a lower bound for the comparison. Many tuples with high Pearson correlation but low mutual information can be detected arising from outlying expression values (Figure 8A). However, pairs of genes with high mutual information and low Pearson correlation, thus indicating a non-linear correlation, are not observed. The only remarkable tuple (marked with an arrow in Figure 7 and shown in Figure 8B) also arises from outlying values. The second dataset contains cDNA measurements for E2 = 102 experiments on G2 = 22608 genes derived from 20 different human tissues [33]. In contrast to the first dataset, tuples with low Pearson correlation but high mutual information are indeed detected. For two exemplary chosen tuples (Figure 8C and 8D), clusters of experimental conditions can be clearly detected by eye. Such type of correlations are missed by analyses based exclusively on linear measures, such as the the analysis done in the original publication of this dataset. For both datasets, tuples calculated from shuffled data (Figure 7, blue data points) result in small values for both similarity measures. Thereby, they indicate a high significance of the original associations. Peaks with high Pearson correlation in the first dataset arise from gene-gene associations with outlying values. Significance values for the exemplarily chosen pairs of genes of the second dataset (Figure 8C, and 8D) were explicitly calculated (Figure 9). They show high significance values for the two examples of observed non-linear correlations on the basis of the mutual information. Compared to this, the significances calculated from the Pearson correlation are poor. In summary, our analysis confirms for the first dataset that the Pearson correlation does not miss any non-linear correlations. As a side effect we are able to detect gene-gene pairs containing outlying values. For the second dataset, however, a substantial amount of non-linear correlations was detected. Gene-gene pairs exemplarily chosen from this fraction show a clustering of data points (experiments) with a high significance. Even though such patterns can be easily found by eye, computational methods need to be applied for the inspection of several hundred million comparisons. Discussion and conclusion After a brief introduction into the information theoretic concept of mutual information, we proposed a method for its estimation from continuous data. Within our approach, we extend the bins of the classical algorithm to polynomial B-spline functions: Data points are no longer assigned to exactly one bin but to several bins simultaneously, with weights given by the B-spline functions. By definition, the weighting coefficients for each data point automatically sum up to unity. Though our algorithm is reminiscent of kernel density estimators [18], it keeps the basic idea to associate data points to discrete bins. In this way, we are able to avoid time-consuming numerical integration steps usually intrinsic to estimates of mutual information using kernel density estimators [20]. To show that our approach improves the simple binning method and to compare it to KDE and the recently reported estimator , we provided a systematic comparison between all these algorithms for artificially generated datasets, drawn from a known distribution. We found that mutual information, as well as its standard deviation, scales linearly with the inverse size of a dataset for the standard binning method, for the B-spline approach, and for . For the KDE approach we find an asymptotic behaviour with a linear tail for large datasets. Moreover, the discrimination of correlations from the hypothesis of statistical independence is significantly improved by extending the standard binning method to B-spline functions, as shown by a two-fold increase of the significance. Compared to KDE, the B-spline functions produce similar significances. However, due to the numerical expenses of the KDE, an application of this algorithm is limited to datasets of mod-erate size. The application of leads to significances in-between the standard binning and the B-spline approach for reasonable bin numbers. Linear correlation measures are among the most applied measures of similarity in the literature. Often, they are used on an ad-hoc basis and it is unclear whether a considerable number of non-linear correlations are missed. Here, we asked the question whether previous analyses, based on linear correlations, sufficiently described the correlations within gene expression datasets or whether mutual information detects additional correlations that are not detected by linear measures, such as the Pearson correlation. For data that is well described by the Pearson correlation, we can give the relation of the Pearson correlation to the mutual information explicitly [32]. Both measures were then applied to publicly available large-scale gene expression datasets [31,33]. We aimed to verify whether non-linear correlations shown as deviations from this relation can be detected. Our findings show that the first dataset is fairly well described by the given relation of the Pearson correlation to the mutual information. No data points with high mutual information and low Pearson correlation are detected. Comparisons of genes containing outlying values, however, result in deviations with low mutual information and high Pearson correlation. From this, it follows that previous analyses on this dataset, based on Pearson correlation, did not miss any non-linear correlations. This presents an important finding since it is by all means supposable that the regulations inherent in the genetic network under consideration might show more complex behaviour than the observed linear ones. Even for one of the largest expression datasets at hand, insufficient data might complicate the detection of such complex patterns of regulation. Alternatively, the biological mechanisms which underlay the regulatory networks might not lead to non-linear correlations. It also has to be considered that the experimental methods applied for the generation of this dataset may make non-linear correlations difficult to detect. The second dataset, in contrast, reveals highly significant tuples with high mutual information and low Pearson correlation. Detailed gene-gene plots of such tuples show that the expression values of the contributing genes fall into groups of experimental conditions. Without attempting to draw conclusions about the biological context of such clusters here, they might reflect interesting situations worth to be analysed in detail. Authors' contributions Most of the manuscript text was written by CD and edited by all authors. CD carried out the calculations and produced the figures. RS strongly contributed to the theoretical background of entropy and mutual information. The implementation of the C++ program was carried out by SK. JS and SK supervised this work. All authors read and approved the final manuscript. Acknowledgements The authors would like to thank Joachim Kopka and Janko Weise for stimulating discussions and Megan McKenzie for editing the manuscript (all of the MPI-MP). RS acknowledges financial support by the HSP-N grant of the the state of Brandenburg. Figures and Tables Figure 1 Two datasets X and Y (100 data points) show a hypothetical dependency f(x) = 4x(1 - x) (top). The Pearson correlation coefficient is not able to detect a significant correlation as shown in the histogram plot of the dataset compared to 300 realisations of shuffled data (left). Mutual information clearly shows that the two datasets are not statistically independent (right). Figure 2 The continuous experimental data for the variable x needs to be binned for the calculation of mutual information. The indicator function of Eq. (5) counts the number of data points within each bin (example with Mx = 5 bins, left). The generalised indicator function based on B-spline functions of Eq. (8) extends the bins to polynomial functions (example with Mx = 5 bins and spline order k = 3, right). The bins now overlap and the weight of each data point to each of the bins is given by the value of the respective B-spline functions at the data point. By definition, all weights contributing to one data point sum up to unity. Figure 3 Mutual information is estimated for artificially generated equidis-tributed random numbers from the simple binning (k = 1), the B-spline approach (k = 3), and the entropy estimator using M = 6 bins, and additionally from the kernel density estimator. The average over an ensemble of 600 trials is shown as a function of the size of the dataset (left) together with the standard deviation (right). Figure 4 Mutual information calculated for a dataset of 300 data points drawn from the distribution shown in Figure 1 (crosses). The number of bins was fixed to M = 6. The average mutual information for 300 shuffled realisations of the dataset is shown (circles) together with the standard deviation as error-bars. The largest value found within the ensemble of shuffled data is drawn as a dotted line (left). The significance was calculated from Eq. (13) (right). Figure 5 Based on the distribution of Figure 1, the mutual information for 300 data points and two spline orders k = 1 and k = 3 is shown as a function of the number of bins M (crosses) together with mean (circles) and standard deviations (error-bars) of 300 surrogates. The dotted lines indicate the largest mutual information found within the ensemble of surrogate data. Figure 6 The significance, S, as a function of the number of bins, M, for the two examples of Figure 5, and for the entropy estimator . For kernel density estimators (KDE), the significance, which is not depending on M, is calculated to S = 92. Figure 7 The Pearson correlation coefficient and the mutual information for all pairwise comparisons of genes for two large-scale gene expression datasets are shown (black points) overlayed by the same measures obtained from shuffled data (blue points). The expected mutual information calculated from Eq. (14) is shown as read curve. For the first dataset (left) genes containing undefined values were omitted resulting in 5345 genes measured under 300 experimental conditions [31]. For the second dataset (right) containing 22608 genes measured under 102 experimental conditions [33], a representative fraction is shown. Figure 8 Examples of gene-gene plots for genes X and Y are shown for characteristic tuples (MI(X,Y), R(X,Y)) detected in Figure (7). For the first gene expression dataset under consideration [31], no non-linear correlations are detected. Moreover, tuples with high Pearson correlation and low mutual information, examples A and B, resulting from outlying values are detected. For the second dataset [33], however, tuples with low Pearson correlation and high mutual information are observed, see examples C and D. Such non-linear correlations are missed by solely using linear correlation measures. Figure 9 Significance values for the two gene-gene comparisons shown in Figure 8, C and D (top and bottom, respectively) are calculated from 300 shuffled realisations based on the Pearson correlation coefficient (left) and the mutual information (right) as distance measures. Table 1 For the calculation of probabilities p(ai) according to the B-spline approach, Mx weighting coefficients are determined for each value xu of variable x. Bi=1,k=2(xu) Bi=2,k=2(xu) Bi=3,k=2(xu) x1 1.0 0.0 0.0 x2 0.6 0.4 0.0 x3 0.2 0.8 0.0 x4 0.0 0.8 0.2 x5 0.0 0.4 0.6 x6 0.0 0.0 1.0 p(ai) 1.8/6 2.4/6 1.8/6 ==== Refs Schena M Shalon D Davis RW Brown PO Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray Science 1995 270 467 470 7569999 Velculescu VE Zhang I Vogelstein B Kinzler K Serial Analysis of Gene Expression Science 1995 270 484 487 7570003 Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns Proc Natl Acad Sci USA 1998 95 14863 14868 9843981 10.1073/pnas.95.25.14863 D'haeseleer P Weng X Fuhrman S Somogyi R Information processing in cells and tissues Plenum Publishing 1997 203 212 D'haeseleer P Liang S Somogyi R Genetic network inference: from co-expression clustering to reverse engineering Bioinformatics 2000 16 707 726 11099257 10.1093/bioinformatics/16.8.707 Michaels GS Carr DB Askenazi M Fuhrmann S Wen X Somogyi R Cluster analysis and data visualization of large-scale gene expression data Pac Symp Biocomput 1998 42 53 9697170 Butte AJ Kohane IS Mutual information relevance networks: functional genomic clustering using pairwise entropy measurements Pac Symp Biocomput 2000 5 427 439 Herwig R Poustka AJ Muller C Bull C Lehrach H O'brien J Large-scale clustering of cDNA-fingerprinting data Genome Res 1999 9 1093 1105 10568749 10.1101/gr.9.11.1093 Korber BT Farber RM Wolpert DH Lapedes AS Covariation of mutations in the V3 loop of human immunodeficiency virus type 1 envelope protein: An information theoretic analysis Proc Natl Acad Sci USA 1993 90 7176 7180 8346232 Gorodkin J Heyer LJ Brunak S Stormo GD Wen X Somogyi R Display the information contents of structural RNA alignments: the structure logos Comput Appl Biosci 1997 13 583 586 9475985 Liang S Fuhrman S Somogyi R Reveal, a general reverse engineering algorithm for inference of genetic network architectures Pac Symp Biocomput 1998 18 29 9697168 Roberts S Everson R Independent component analysis: Priciples and Practice 2001 Cambridge: Cambridge University Press Hyvärinen A Karhunne J Oja E Independent component analysis 2001 New York: Wiley Fraser AM Swinney HL Independent coordinates for strange attractors from mutual information Phys Rev A 1986 33 2318 2321 10.1103/PhysRevA.33.1134 Thénevaz P Unser M Optimization of mutual information for multiresolution image registration IEEE Trans Image Processing 2000 9 2083 2099 10.1109/83.887976 Ellis DP Bilmes JA Using mutual information to design feature combinations In Proceedings of the International Conference on Spoken Language Processing: Beijing 16–20 October 2000 Shannon CE A mathematical theory of communication The Bell System Technical Journal 1948 27 623 656 Moon Y Rajagopalan B Lall U Estimation of mutual information using kernel density estimators Phys Rev E 1995 52 2318 2321 10.1103/PhysRevE.52.2318 Silverman BW Density estimation for statistics and data analysis 1986 London: Chapman and Hall Steuer R Kurths J Daub CO Weise J Selbig J The mutual information: detecting end evaluating dependencies between variables Bioinformatics 2002 S231 S240 12386007 Paninski L Estimation of Entropy and Mutual Information Neural Computation 2003 15 1191 1253 10.1162/089976603321780272 DeBoor C A practical guide to splines 1978 New York: Springer Unser M Aldroubi A Eden M B-spline signal processing: Part 1 – Theory IEEE Trans Signal Precessing 1993 41 821 832 10.1109/78.193220 Unser M Aldroubi A Eden M B-spline signal processing: Part 2 – Efficient design and applications IEEE Trans Signal Precessing 1993 41 834 848 10.1109/78.193221 Herzel H Schmidt AO Ebeling W Finite sample effects in sequence analysis Chaos, Solitons & Fractals 1994 4 97 113 10.1016/0960-0779(94)90020-5 Herzel H Grosse I Measuring correlations in symbol sequences Physica A 1995 216 518 542 10.1016/0378-4371(95)00104-F Grosse I Freund JA Estimating entropies from finite samples In Dynamik, Evolution, Strukturen 1996 Berlin: Dr. Köster 181 190 Roulston MS Estimating the error on measured entropy and mutual information Physica D 1999 125 285 294 10.1016/S0167-2789(98)00269-3 Herzel H Grosse I Correlations in DNA sequences: The role of protein coding segments Phy Rev E 1997 55 800 810 10.1103/PhysRevE.55.800 Klus GT Song A Schick A Wahde M Szallasi Z Mutual Information Analysis as a Tool to Assess the Role of Aneuploidy in the Generation of Cancer-Associated Differential Gene Expression Patterns Pac Symp Biocomput 2001 42 51 11262960 Hughes TR Marton MJ Jones AR Roberts CJ Stoughton R Armour CD Bennett HA Coffey HA Dai H He YD Kidd MJ King AM Meyer MR Slade D Lum PY Stepaniants SB Shoemaker DD Gachotte D Chakraburtty K Simon J Bard M Friend SH Functional Discovery via a Compendium of Expression Profiles Cell 2000 102 109 126 10929718 10.1016/S0092-8674(00)00015-5 Steuer R Daub CO Selbig J Kurths J Measuring distances between variables by mutual information In Proceedings of the 27th Annual Conference of the Gesellschaft für Klassifikation: Cottbus 12–14 March 2003 He YD Dai H Schadt EE Cavet G Edwards SW Stepaniants SB Duenwald S Kleinhanz R Jones AR Shoemaker DD Stoughton RB Microarray standard data set and figures of merit for comparing data processing methods and experiment design Bioinformatics 2003 19 956 965 12761058 10.1093/bioinformatics/btg126

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-261528578610.1186/1471-2202-5-26Research ArticleSlowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi Dozmorov Mikhail [email protected] Rui [email protected] Hui-Ping [email protected] Barbro [email protected]öm Holger [email protected] Department of Medical Biophysics, Institute of Physiology and Pharmacology, Göteborg University, Box 433, SE 405 30 Göteborg, Sweden2004 3 8 2004 5 26 26 10 5 2004 3 8 2004 Copyright © 2004 Dozmorov et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through periods of receptor blockade. The similarity to key features of long-term depression and long-term potentiation suggests a possible relation to these phenomena. Additionally, a short term potentiation and decay (<5 min) were observed during sudden start of NMDA receptor activation supporting the idea that NMDA receptor mediated responses are highly plastic. ==== Body Background Hippocampal synapses display a variety of activity dependent changes that may represent basic elements of memory. Of foremost interest are long-term potentiation (LTP) and depression (LTD), especially forms that depend on N-methyl-D-aspartate (NMDA) receptor activation and therefore can attain "associative" properties [1-3]. The selective induction of LTP versus LTD has been attributed to differing amounts of Ca2+ ions entering via postsynaptic NMDA receptor channels [4]. Depending on type of stimulation, enzymes with different sensitivities to Ca2+ may be engaged and change the balance between kinase and phosphatase activities, leading to either phosphorylation or dephosphorylation of postsynaptic target proteins, such as ionotropic receptors [2]. It has been shown that afferent stimulation by frequencies in the range 0.5 to 5 Hz reliably produces LTD whereas higher frequencies, 50–100 Hz, lead to LTP [5]. Several studies suggest that temporal factors are also important, implying that LTD requires a longer time to be induced than LTP [6]. We have previously demonstrated that under conditions of facilitated activation of NMDA receptors by low extracellular Mg2+ synaptic plasticity can be induced by frequencies as low as 0.1–0.2 Hz when applied for prolonged periods of time [7]. Following an initial phase of transient potentiation there was a substantial depression that developed gradually during several hours and that remained stable after termination of NMDA receptor activation. Although the relation to "standard LTD" was not fully clarified, such slowly developing depression in low Mg2+ solution may provide a useful model for studying certain forms of NMDA receptor dependent depression. In the present study, we will further develop the concept of gradually decaying responses. One critical issue regarding LTP, LTD as well as other forms of glutamatergic synaptic plasticity, is the relative contribution of different glutamate receptor subtypes in creating the synaptic modification. Knowledge about this matter may be helpful in elucidating the underlying modification. While a selective change of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been cherished [8-10], especially in the case of LTP, several studies also observed NMDA receptor mediated changes in both LTP and LTD [11-14]. Previous work on LTD in our lab described an equal change of AMPA and NMDA responses [15]. However, it was reported by others that the relative contributions of AMPA and NMDA responses during LTD depend on experimental conditions, an equal change being one possible outcome [12]. In our recent examination of a slowly developing depression using composite AMPA-NMDA excitatory postsynaptic potentials (EPSPs) [7], the two responses declined in close parallel, indicating a common factor. Such an equal change is compatible with both a coordinated change of receptors and a presynaptic one via a decrease of glutamate release. However, in view of other studies reporting a coupling between responses via AMPA and NMDA receptors [16,17], one may ask whether our observation of declining NMDA responses could be secondary to the change of AMPA. In the present study, isolated NMDA receptor mediated EPSPs were shown to decline progressively during prolonged low frequency activation (0.1 Hz). Moreover, following sudden start of stimulation there was an initial, transient potentiation. Our findings also resolved some questions regarding input specificity and durability of the slow decay, which were previously addressed only for AMPA EPSPs. Results Isolated NMDA EPSPs show a progressive decay AMPA EPSPs were initially recorded in low Mg2+ solution in the presence of NMDA receptor antagonist AP5 to allow for pathway equalization (see Methods) without evoking NMDA EPSPs. Synaptic transmission was then entirely blocked by adding AMPA receptor antagonist CNQX, followed by unblocking of NMDA receptors by wash out of AP5. During this time, only one pathway was stimulated, keeping the other one silent for later use. As illustrated in Fig. 1A (upper part), an NMDA receptor mediated EPSP appeared within 10 min and reached maximum about 30 min after switching to AP5-free solution. During the following recording period of nearly 2 h, the NMDA EPSP decayed substantially, on average down to 58 ± 6 % of peak value (n = 8). Control experiments showed that isolated AMPA EPSPs recorded in low Mg2+ remained stable for several hours (changing to 96 ± 5 % of baseline after 2 h, n = 5, not illustrated). Reinduction in a naive pathway To exclude the possibility that the observed decay of NMDA responses were due to deterioration of slices, implying a general decrease of essential physiological processes, the experiment was repeated for the other pathway, i.e. the one that had not previously expressed NMDA responses (continuation in the same set of 8 slices). As seen in Fig. 1A (lower part), a similar result was obtained as above. The NMDA EPSP peaked at 98 ± 7 % and declined to 63 ± 8 % relative to the peak in the first experimental session. As illustrated in Fig. 1B, the curve obtained for a naive pathway during the second session of NMDA unblocking was similar to the one obtained for the pathway activated during the first session, the two curves overlapping closely for the entire recording period. Comparison between pathways Specificity and persistency The same experiment was used to address the question of persistency as well as input specificity of the slowly developing depression of NMDA EPSPs. As can be seen in Fig. 1A, the pathway that was active during the first experimental session was retested during the second one together with the pathway receiving novel NMDA receptor activation. This allowed for a comparison between the pathways (for convenience, the peak in the first session is still used as reference for the values in the following). During the second session of NMDA receptor unblocking, the previously treated pathway displayed a substantially smaller peak than the naive one (48 ± 3 % vs. 98 ± 7 % p < 0.05, n = 8), and the two curves were still different by the end of the session (44 ± 3 % vs. 63 ± 8 %; difference 19 ± 4 %, p < 0.05). Since the latter time point was located at 3 h after the end of the first session it is evident that the depression of NMDA EPSPs lasted for at least 3 h. It is noteworthy that the previously depressed pathway showed no significant decay during the second session, passing from 48 to 44 %, i.e. a relative change by 92 % (p > 0.05), as if it had already been saturated. For a comparison, the naive pathway changed by a factor 63/98 = 64 % (p < 0.05) (see curves in Fig. 1C). A graphic summary of all "peak" and "end-of-session" values is given in Fig. 3B. It can also be noted that NMDA EPSPs recorded for contiguous time intervals up to 4 h reached a saturation level after 2–3 h (n = 3, not illustrated). Instantaneous versus persistent depression The above results suggest that the progressive decay observed in a single pathway as an instant event actually represents a long-term, pathway-specific change that can be assessed long later by comparison across pathways. To further examine the relation between the instantaneously recorded depression and the one measured about 90 min later, the relation between the two was plotted as illustrated in Fig. 3C. The two variables were found to be positively correlated (r = 0.71, p < 0.05, n = 8), implying that depression to a lower level in one pathway led to smaller responses in that pathway at later times compared to another pathway. The regression line, passing below rather than through the point of no depression (100 %, 100 %), indicates that even a slight instantaneous decay may be coupled to a noticeable change in the long term. Possibly, the declining trend was partially masked by recovery from AP5 leading to an underestimation of it. Effect of NMDA receptor blockade on subsequent NMDA EPSP decay To pinpoint the induction mechanism, in terms of a pre- versus postsynaptic location, experiments were carried out in a similar way as above except for keeping AP5 in the solution during the first session (see Fig. 2A). Hence, the stimulus pattern included a 3 h long interval with no stimulation in one of the pathways. The other pathway was stimulated during that time, and most likely releasing glutamate, but no postsynaptic response was expressed due to blockade of NMDA receptors. It can be argued that successful blockade of depression would predict a postsynaptic mechanism whereas a failure to do so would predict a presynaptic one. Fig. 2B shows that the depression was actually blocked, demonstrating the importance of NMDA receptor activation in the induction process. As illustrated, the two curves obtained during unblocking of NMDA receptors in the second session were quite similar. The continuously stimulated pathway, being depressed in the standard case, peaked at a level of 105 ± 5 % relative to the control pathway (n = 5; see also graphic summary of values in Fig. 3B). Phase trajectories as indicators of waveform change Depending on the type of synaptic modification, EPSP waveforms may change in different ways, and previous work in our lab has demonstrated that NMDA EPSPs are more prone than AMPA EPSPs to show these changes [18]. Taking advantage of such waveform analysis, which might shed light on the underlying mechanism, we examined phase plots based on measures of the initial part and the later part of the NMDA EPSP (see Methods) on the X- and Y-axis, respectively. The curve in Fig. 3D is based on a total of 20 experiments (pathways), including some with only a single session. As shown, the phase trajectory displayed a loop indicating a difference between the effect of AP5 and the gradual decay of NMDA EPSPs. On the average the encircled area was 15 ± 2 % (p < 0.05, n = 20; scaling peak × peak as 100 %, clockwise being positive). While our data show that the time window matters for measuring NMDA EPSPs, all the above results were qualitatively similar regardless of which window was used. It can be noted, however, that the depression of NMDA EPSPs by the end of a recording session was less pronounced using early measurements than late ones (responses attaining 72 ± 3 % vs. 61 ± 3 % of the peak value, p < 0.05, n = 20). In 12 out of the 20 experiments, the fiber volley was well separated from the stimulus artifact allowing it to be properly measured. No significant change was detected, the end-of session value amounting 103 ± 2 % of the value at the EPSP peak (p > 0.05, not illustrated). Short-term effects induced by sudden onset stimulation In the above, NMDA receptor activation occurred gradually while the antagonist AP5 was washed out. This is in line with the experimental protocol used in our previous work on composite EPSPs containing both AMPA and NMDA components [7]. However, a natural question is whether sudden, novel activation of NMDA receptors is equivalent in producing the results observed here. We therefore pursued experiments in which stimulation was silenced until washout of AP5 was complete. One pathway, receiving such sudden stimulation, was compared to a control pathway subjected to gradual NMDA receptor activation during a single recording session. Fig. 4A shows an essential difference in behavior between the pathways, the sudden start of activation leading to substantially larger responses for about 5 min. Fig. 4B reveals additional complexity, the initial responses showing actual growth of responses for about a minute before they started to decay, implying an early potentiation process. The total range of responses was substantial, from a peak above 200 % to about 70 % by the end of the recording session (relative to the peak of the control pathway), i.e. about 3 times. In order to determine whether the transient potentiation had any obvious relation to the slow depression of NMDA EPSPs, the relation between the two was examined. Thus, the degree of initial potentiation was calculated by comparing the pathways just after stimulation was started and the depression was determined, as before, by comparing the end-of-session value with the peak value (see legend of Fig. 4 for further details). The two variables, illustrated by the XY-plot in Fig. 4C, were found to have no significant correlation (r = 0.35, p > 0.05). Discussion Our study revealed a progressive decline of pharmacologically isolated NMDA EPSPs, as observed for several hours in response to low rate (0.1 Hz) activation of afferents. The decline was found to be a form of long-term synaptic depression with an induction linked to NMDA receptor activation and with an expression that was maintained through periods without such activation. Several of its basic characteristics were similar to those of conventional LTP and LTD, suggesting a possible relation to these phenomena. Synapse specificity and NMDA-dependent induction Decaying responses is a potential side effect in long-term, electric recording in vitro due to declining viability of biological tissue or other experimental imperfections. Such unspecific "run down" can not account for the present findings since the gradual depression of responses could be repeated in the same slice, using a previously undepressed pathway. On the other hand, if the experiment was repeated twice in the same pathway, the second occasion revealed a diminished NMDA EPSP that showed little further decay. Together, these results show that the depression is input specific and long lasting and that it can saturate. Moreover, the lack of associated changes of the fiber volley speaks against a failure of axon conductance [19], favoring a synaptic localization of the process. While both pre- and postsynaptic expression mechanisms appear feasible, certain mechanisms of induction can be excluded. For instance, a decrease in probability of glutamate release due to a direct depletion of the vesicle pool is unlikely since AMPA EPSPs could be evoked for several hours without significant decay (see also [7,20]). Even so, a use-dependent reduction of vesicle content may affect NMDA responses selectively under certain conditions by restricting "glutamate spillover" [21]. The most critical data with respect to the induction mechanism is that a period of conditioning stimulation, normally leading to reduction of NMDA EPSPs in the same pathway later on, was ineffective if delivered during blockade of NMDA receptors. This implies that the induction of the depression requires activation of NMDA receptors, most likely postsynaptically. Other observations of decaying NMDA EPSPs The input specificity and NMDA dependent induction of the current depression conform with basic properties of conventional LTP and LTD [22]. The depression might then be a case of LTD, although induced by an alternative protocol. In fact, LTD was shown to be associated with changes involving both AMPA and NMDA receptors, although the linkage between the two contributions is controversial [12,15]. Moreover, both of the cited studies demonstrated LTD of isolated NMDA EPSPs induced by 1–2 Hz stimulation. In contrast, experiments in cultures, inducing LTD by field stimulation at a higher frequency (5 Hz), reported only AMPA receptor mediated changes [10]. Direct interaction tests may further clarify the relation between the present depression and LTD. Gradually decaying, NMDA receptor mediated responses were observed previously in our lab during recording of composite AMPA-NMDA EPSPs for several hours [7,23]. Attempts to relate the decay to LTD demonstrated a weak reduction of subsequent LTD of AMPA responses suggesting at least some elements in common [7]. In view of studies reporting forms of AMPA-NMDA coupling [16,17], it is arguable that the studies demonstrating a decay of both components could have been influenced by the use of composite responses. In one of our studies [7], the observed depression of the AMPA component of composite EPSPs was verified by additional comparison between isolated AMPA EPSPs obtained under blockade of NMDA receptors. A similar verification was lacking for the depression of the NMDA response. By recording of isolated NMDA EPSPs, the present study ascertains that NMDA receptor mediated responses undergo a use-dependent depression, which is manifested in the absence of AMPA receptor activation. However, the decay was less pronounced than that reported previously for the NMDA component of composite EPSPs (average reduction to 60 % of peak as compared to 40 % in the previous study [7]). While we observed that isolated NMDA EPSPs decay "spontaneously", most prior studies employing such EPSPs did not report a decay. It might be that limitations of recording time concealed the effect and cell dialysis during whole cell recording could also be a limiting factor. Actually, a recent study, recording "novel" responses under whole cell conditions, reported on decaying AMPA EPSPs but constant NMDA EPSPs [24]. The possibility of AMPA receptor LTD under the present conditions could not be excluded as the blockade of the receptors may just conceal the effect. Further studies may help to reveal this matter. Persistency and saturability Standard LTP/LTD experiments compare relatively stable periods of recording before and after induction of the synaptic modification. This was not possible in the present case, since merely test stimulation evoked the decay. Therefore, comparisons were generally made between synaptic pathways subjected to different stimulus paradigms. The induction of depression in a single pathway during an initial 2 h period caused a subsequent difference between NMDA responses of the two pathways throughout a subsequent test period. The degree of initial decay was closely related to the later difference between pathways, suggesting that once depression occurred it could be maintained through periods of receptor blockade until testing was performed. Our data suggest a duration of the depression of more than 3 h after the initial induction period. This is in the range commonly referred to as "late", which is believed to involve special biochemistry such as gene expression and protein synthesis [25,26]. Whether, the presently studied depression involves such changes remains to be determined. The gradual depression of NMDA EPSPs was found to saturate after 2–3 h as evidenced by both single and double session experiments. This is in line with several other forms of NMDA-dependent plasticity, including LTP, LTD and chemically induced variants, which are shown to be saturable [27-29]. Whether, the saturation observed here is a "true one" at the level of expression is not known. Alternatively it could be a phenomenon at the induction level, related to weaker induction due to the diminished NMDA response. Possible expression mechanisms Previous work on conventionally induced LTD revealed an essential role for protein phosphatases in mediating the synaptic modification [30,31]. Consistent with the idea that changes of AMPA receptors mediate NMDA-dependent synaptic plasticity [32,33] it was demonstrated that certain sites of the GluR1 subunit were targeted in LTP/de-potentiation and other ones in LTD/de-depression [2,34]. Less is known about mechanisms underlying NMDA receptor changes in LTP/LTD as well as in the current depression. A previous study in our lab recording composite EPSPs reported that LTD of the NMDA component was blocked by a phosphatase inhibitor in a similar manner as "standard LTD" [15]. Hence, one can envisage that NMDA receptors would be controlled via dephosphorylation in a similar manner as inferred for AMPA receptors. NMDA receptors also have a number of other regulatory sites, allowing for modulation by glycine, polyamines, calcium, and redox agents [35] and they have shown to be mobile as well [36-38], in keeping with the idea of mobile AMPA receptors in LTP/LTD [39,40]. Regardless of details, additional factors are needed to stabilize the synaptic modification in the long term, perhaps via synthesis of new proteins as previously demonstrated for LTP and LTD lasting longer than about 3 h [25,28,41]. Changes in synaptic morphology and altered subunit composition of receptors are examples of protein synthesis dependent mechanisms that have been implied in late forms of plasticity [32,42]. Although a postsynaptic modification appears to be the primary choice, a presynaptic one that is initiated postsynaptically is also conceivable. In previous attempts to distinguish between pre- and postsynaptic mechanisms, LTD was compared with depression caused by various pharmacological agents with respect to the ability to influence the waveform of EPSPs [18]. While LTD in that study was found to affect isolated NMDA EPSPs in a uniform manner, i.e. no waveform change, the present data appeared to be less clear-cut. Nevertheless, the relation between early and late EPSP measurements differed for the initial AP5 washout period and the following period of actively induced depression, indicating a change in EPSP waveform. The depression therefore appeared to be distinct from a postsynaptic modification via modulation of channel gating. However, a clear test of the pre-post issue still remains. Unfortunately, the MK-801 test of release probability [43] does not appear useful when dealing with decaying responses as in the present case. Short-term changes and their possible mechanisms While the main line of experiments employed a smooth start of NMDA receptor activation following the gradual washout of AP5, another set of experiments made use of sudden activation by awaiting full washout until stimulation was started. Compared to smooth activation, there was an additional, transient potentiation that largely decayed within 20–30 stimuli. This is in accord with a previous study in hippocampal slices showing that stopping stimulation of composite AMPA-NMDA EPSPs for 10–60 min (and one case of isolated NMDA EPSP for 10 min) resulted in a transient potentiation when stimulation was resumed [23]. Several other studies describe decaying NMDA responses in relation to inactivation or desensitization of receptors [44,45]. Accordingly, synaptically evoked NMDA responses in cell cultures were found to inactivate (i.e. decay) within a few minutes in much the same manner as observed here [45], a process shown to be triggered by postsynaptic influx of Ca2+ via the NMDA channels. Similar mechanisms of receptor desensitization/inactivation might be responsible in the present case in forming the transient phase after starting stimulation. Some details remain unexplained by this simple model, such as the biphasic character of the transient phase in terms of initial growth and subsequent decay. One can speculate that a minor LTP, or short-term potentiation, might be induced by the sudden activation of NMDA receptors and so would contribute to the initial growth, although the underlying cause is not addressed in this kind of explanation. Conclusions The above results emphasize that NMDA receptor mediated responses are highly plastic and that mere test stimulation can induce a short-term potentiation as well as a slowly developing depression that persists for several hours. The depression was input specific and saturable, and its induction required NMDA but not AMPA receptor activation in conformity with conventionally induced LTP and LTD, suggesting a relation to these phenomena. While a low Mg2+ solution was used in our case to unblock NMDA receptors, similar unblocking may occur naturally in response to depolarization. Several important issues are still not settled. Is the saturation of the NMDA EPSP depression an absolute matter or can it be overcome, leading to further down regulation and possibly silencing of synapses? Conversely, is it possible to reverse, i.e. de-depress, the change by LTP or similar processes, allowing for bidirectional control? Further research is needed to resolve these questions. Methods Experiments were performed on 12 to 18 day old Sprague-Dawley rats. The animals were decapitated after isoflurane (Forene) anesthesia in accordance with the guidelines of the Swedish Council for Laboratory Animals. All animal procedures were approved by the Local Ethics Committee at Göteborg University. The brain was removed and placed in an ice-cold artificial cerebrospinal fluid solution containing (in mM) NaCl 119, KCl 2.5, CaCl2 2, MgCl2 2, NaHCO3 26, NaH2PO4 1 and glucose 10, oxygenated by 95% O2, 5% CO2. The hippocampus was dissected out and transverse 400 μm thick slices were prepared by a vibratome or tissue chopper. The slices were initially kept in the same solution at room temperature for at least 60 min. As required, slices were then transferred to one or several "submerged type" recording chambers. During the experiment, slices were perfused at 30°C by a solution similar to that above except that the concentration of Mg2+ was 0.1 mM. The usage of low Mg2+ allowed for expression of NMDA receptor mediated responses. Stimulation was delivered as 0.1 ms negative constant current pulses via monopolar tungsten electrodes. For each slice, two stimulating electrodes were placed in the apical dendritic layer of CA1 pyramidal cells on either side of the recording electrode to provide for stimulation of two separate sets of afferents. Field EPSPs were recorded by using a glass micropipette filled with 3 M NaCl (4–10 MΩ resistance). The basal test stimulus frequency was 0.1 Hz with stimuli delivered alternately to the two electrodes, successive stimuli being separated by 5 s. To test the effect of stimulus interruption, one of the two electrodes was given no stimulation during a certain time, the other one remaining stimulated at 0.1 Hz. Recording commenced by monitoring isolated AMPA EPSPs in the presence of AP5 (50 μM) to block NMDA responses. A low concentration of CNQX (1 μM) was used to partially suppress the AMPA responses. In this way, somewhat larger stimulus strengths could be applied, suitable for evoking isolated NMDA EPSPs in the later part of the experiment. During the time of AMPA EPSP recording, the stimulus strengths were adjusted for each slice to equalize the synaptic inputs of the two pathways. This was essential for later comparison of NMDA EPSP across pathways. After obtaining a baseline of equal AMPA responses, the concentration of CNQX was raised to 10 μM which entirely blocked synaptic responces. The remaining non-synaptic response, consisting of stimulus artifact and presynaptic volley, was used to define "true zero". To study NMDA receptor mediated responses, CNQX (10 μM) was maintained in the solution while AP5 was washed out for one or several 2 h periods, referred to as sessions in the following. In between the sessions as well as afterwards, synaptic transmission was again blocked by applying AP5 (50 μM), framing in the sessions by periods of recording non-synaptic responses. Under the sessions, various tests were made depending on the purpose of investigation. Usually one input remained silent during the first session and stimulation was not resumed until after synaptic transmission was reblocked. In another kind of experiment, the initially silent pathway was reactivated in the early part of the first session after NMDA receptors were unblocked, providing a means for sudden start of NMDA receptor activation. Signals were amplified, filtered and transferred to a PC clone computer for on-line and off-line analysis by specially designed electronic equipment (based on an Eagle Instruments multifunction board) and own developed computer software. AMPA EPSPs were measured using an early time window (first 1.5 ms after the fiber volley) while NMDA EPSPs were measured using both an early (first 5 ms after volley) and a late (35–45 ms after artifact) time window. The late measurement was used in presenting most of the results, allowing easy comparison with previous work in our lab that estimated the NMDA component of composite EPSPs via a late measurement [7]. Similar albeit not identical results were obtained with early and late measurements (see illustration in Fig. 3D). Measurements were calculated by integrating the curve along the specified time window after substraction of the prestimulus baseline. All values were corrected by substracting the corresponding measurements of the non-synaptic potential obtained after total blockage of the EPSPs (except when measuring the fiber volley). The final data were quantified as relative values compared to a reference level defining 100 %. While the initial baseline formed a natural reference for AMPA responses, the choice was less obvious for NMDA responses, leading us to use the highest level of responses for one of the pathways in one of the experimental sessions (selected to make sense). Results are expressed as mean ± S.E.M. Statistical comparisons were made using Student's t-test. Drugs were obtained from Tocris Cookson, UK; prefabricated stimulating electrodes were obtained from World Precision Instruments, FL USA, type TM33B. Abbreviations AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AP5, D(-)-2-amino-5-phosphonopentanoic acid; CA, cornu ammonis; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EPSP, excitatory postsynaptic potential; LTD, long-term depression; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate; Authors' contributions MD planned and carried out most of the experiments including data analysis, and compiled the manuscript. RL carried out experiments, participated in the planning process and helped in shaping the final manuscript. HPX carried out the initial experiments establishing the effect of NMDA EPSP depression. BJ was responsible for logistics planning and participated in experiments. HW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements This study was supported by the Swedish R.C./medicine 05954 and the foundations of Adlerbert, Åhlen, Märtha & Gustaf Ågren, Herbert & Karin Jacobsson, Sigurd & Elsa Golje, Wilhelm & Martina Lundgren, Svenska Läkaresällskapet, Göteborgs Läkaresällskap, and Alzheimerfonden. Figures and Tables Figure 1 Decay of NMDA receptor mediated EPSPs in low Mg2+ solution. (A) Experimental design: Measurements of field EPSPs from a representative experiment are plotted for two independent pathways, referred to as input 1 and input 2. By appropriate use of specific blockers CNQX and AP5, either isolated AMPA EPSPs (used for initial pathway equalization) or isolated NMDA EPSPs (used in testing sessions N1 and N2) were recorded with periods of fully blocked responses in between. The pathways were stimulated alternately, each at 0.1 Hz, except for silencing input 2 for a 3 h period that contained session N1. Each point represents the average of measurements within 1 min. As seen, during N1 the responses to input 1 decayed. During session N2 both inputs were stimulated, revealing a novel decay for input 2 and occluded depression for input 1. Samples of recorded potentials of both inputs are given for the indicated time points a-e. (B) Superimposed, averaged time courses of NMDA EPSPs for input 1 during session N1, and input 2 during session N2. (C) Superimposed averaged time courses of NMDA EPSPs for input 1 and input 2, both during session N2. For B and C, each point represents the average of measurements within 3 min intervals. The peak during session N1 was used as 100 %. Values are expressed as mean ± S.E.M (n = 8 experiments). Figure 2 Activation of NMDA receptors is necessary for inducing persistent depression. (A) Experimental design: Measurements of field EPSPs are plotted for a representative experiment as in Fig. 1A. The experiment conformed with the previous one except that AP5 was present in the solution during session N1. Accordingly, synaptic transmission was blocked for the entire period when input 2 was stopped. In session N2, AP5 was eventually washed out and both inputs displayed decaying NMDA EPSPs. Samples of recorded potentials of both inputs are given for the indicated time points a-e. (B) Superimposed averaged time courses of NMDA EPSPs from both inputs during session N2. Each point represents the average of measurements within 3 min intervals. The peak of input 2 was chosen as 100 %. The equal appearance of the curves shows that no pathway specific depression remained during N2 due to the differential treatment during N1 (compare such a depression in the reference case in Fig. 1C). Values are expressed as mean ± S.E.M (n = 5 experiments). Figure 3 Summary of NMDA EPSP depressions during unblocking of NMDA receptors. (A1) Schematic representation of the experiment described in Fig. 1. (A2) Schematic representation of the experiment described in Fig. 2. Pathways are defined as "treated" or "naive" depending on whether stimulation was "on" (solid) or "off" (dashed) during session N1. Each number (1–10) in A1 and A2 represents a portion where measurements were taken for analysis. (B) Comparison between NMDA EPSP measurements, each bar representing an average obtained for 30 consecutive responses (5 min). Value no. 1 was chosen as 100 % for experiments depicted in A1. Since a corresponding value was lacking for experiments depicted in A2, value no. 9 was chosen as 100 %. All values are expressed as mean and S.E.M (n = 8 and n = 5, respectively, for A1 and A2 type experiments). (C) Relation between instantaneous depression in session N1 (abscissa, final value relative to peak) and persistent depression in session N2 (ordinate, interpathway comparison of peaks). Each dot represents a single experiment. The analysis revealed a significant correlation between the variables as illustrated by the superimposed regression line (n = 8). (D) Relation between early and late measurements obtained with two different time windows (initial 5 ms and 35–45 ms, respectively) plotted as a "phase trajectory" (abscissa, early measure; ordinate, late measure). The curve represents an average of 20 experiments with additional smoothing to reduce noise (5 min moving average). Samples of recorded NMDA EPSPs corresponding to locations a, b of the trajectory are overlaid. Bars above EPSPs curves indicate time windows used for early and late measurements, respectively. The curve at b is shown superimposed on a dotted copy of the curve at a. Figure 4 Transient potentiation induced by sudden activation of NMDA receptors. (A) Superimposed averaged time courses of NMDA EPSPs for two pathways subjected to different stimulation protocols. Each point represents the average of measurements within 3 min intervals. Stimulation of one pathway (input 2) was stopped for a period of 30 min and then restarted at a time point when NMDA receptors were fully unblocked as judged by responses for the other pathway (input 1) that was exposed to the standard protocol. The peak of input 1 was taken as 100 %. (B) Time course of the newly evoked NMDA responses. Expanded portion of A immediately following restart of stimulation, each point representing measurement of a single EPSP. It can be noted that following the initial response there was additional growth for about a minute before responses started to decay. Values in both A and B are expressed as mean ± S.E.M (n = 8 experiments). (C) Examining the relation between the slow depression and the transient potentiation of NMDA EPSPs. Depression was quantified as end-of-session value relative to peak value (5 min averages) for input 1 that received the standard protocol. Transient potentiation was estimated as the average of the first 18 responses (3 min) of input 2 relative to the average of the corresponding interleaved responses of input 1. The latter value was nearly the same as the peak value. Each dot represents a single experiment (same set of 8 experiments as in A, B). The analysis revealed no significant correlation between the data points. ==== Refs Bliss TV Collingridge GL A synaptic model of memory: long-term potentiation in the hippocampus Nature 1993 361 31 39 8421494 10.1038/361031a0 Malenka RC Nicoll RA Long-term potentiation–a decade of progress? Science 1999 285 1870 1874 10489359 10.1126/science.285.5435.1870 Gustafsson B Wigström H Physiological mechanisms underlying long-term potentiation Trends Neurosci 1988 11 156 162 2469184 10.1016/0166-2236(88)90142-7 Yang SN Tang YG Zucker RS Selective induction of LTP and LTD by postsynaptic [Ca2+]i elevation J Neurophysiol 1999 81 781 787 10036277 Dudek SM Bear MF Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade Proc Natl Acad Sci U S A 1992 89 4363 4367 1350090 Mizuno T Kanazawa I Sakurai M Differential induction of LTP and LTD is not determined solely by instantaneous calcium concentration: an essential involvement of a temporal factor Eur J Neurosci 2001 14 701 708 11556894 10.1046/j.0953-816x.2001.01679.x Dozmorov M Niu YP Xu HP Xiao MY Li R Sandberg M Wigström H Active decay of composite excitatory postsynaptic potentials in hippocampal slices from young rats Brain Res 2003 973 44 55 12729952 10.1016/S0006-8993(03)02536-8 Durand GM Kovalchuk Y Konnerth A Long-term potentiation and functional synapse induction in developing hippocampus Nature 1996 381 71 75 8609991 10.1038/381071a0 Kauer JA Malenka RC Nicoll RA A persistent postsynaptic modification mediates long-term potentiation in the hippocampus Neuron 1988 1 911 917 2908443 Carroll RC Lissin DV von Zastrow M Nicoll RA Malenka RC Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures Nat Neurosci 1999 2 454 460 10321250 10.1038/8123 Clark KA Collingridge GL Synaptic potentiation of dual-component excitatory postsynaptic currents in the rat hippocampus J Physiol 1995 482 39 52 7730988 Selig DK Hjelmstad GO Herron C Nicoll RA Malenka RC Independent mechanisms for long-term depression of AMPA and NMDA responses Neuron 1995 15 417 426 7544143 10.1016/0896-6273(95)90045-4 Muller D Lynch G Long-term potentiation differentially affects two components of synaptic responses in hippocampus Proc Natl Acad Sci U S A 1988 85 9346 9350 2904150 Asztely F Wigström H Gustafsson B The Relative Contribution of NMDA Receptor Channels in the Expression of Long-term Potentiation in the Hippocampal CA1 Region Eur J Neurosci 1992 4 681 690 12106312 Xiao MY Karpefors M Gustafsson B Wigström H On the linkage between AMPA and NMDA receptor-mediated EPSPs in homosynaptic long-term depression in the hippocampal CA1 region of young rats J Neurosci 1995 15 4496 4506 7540677 Bazhenov AV Kleshchevnikov AM Reciprocal inhibition of the AMPA and NMDA components of excitatory postsynaptic potentials in field CA1 of the rat hippocampus in vitro Neurosci Behav Physiol 1999 29 719 725 10651331 Watt AJ van Rossum MC MacLeod KM Nelson SB Turrigiano GG Activity coregulates quantal AMPA and NMDA currents at neocortical synapses Neuron 2000 26 659 670 10896161 10.1016/S0896-6273(00)81202-7 Xiao MY Niu YP Wigström H Comparing long-term depression with pharmacologically induced synaptic attenuations in young rat hippocampi Synapse 1997 26 329 340 9215592 10.1002/(SICI)1098-2396(199708)26:4<329::AID-SYN1>3.3.CO;2-U Thinschmidt JS Walker DW King MA Chronic ethanol treatment reduces the magnitude of hippocampal LTD in the adult rat Synapse 2003 48 189 197 12687638 10.1002/syn.10203 Xiao MY Niu YP Wigström H Activity-dependent decay of early LTP revealed by dual EPSP recording in hippocampal slices from young rats Eur J Neurosci 1996 8 1916 1923 8921282 Kullmann DM Asztely F Extrasynaptic glutamate spillover in the hippocampus: evidence and implications Trends Neurosci 1998 21 8 14 9464678 10.1016/S0166-2236(97)01150-8 Bear MF Malenka RC Synaptic plasticity: LTP and LTD Curr Opin Neurobiol 1994 4 389 399 7919934 10.1016/0959-4388(94)90101-5 Niu YP Xiao MY Karpefors M Wigström H Potentiation and depression following stimulus interruption in young rat hippocampi Neuroreport 1999 10 919 923 10321460 Xiao MY Wasling P Hanse E Gustafsson B Creation of AMPA-silent synapses in the neonatal hippocampus Nat Neurosci 2004 7 236 243 14966524 10.1038/nn1196 Sajikumar S Frey JU Anisomycin inhibits the late maintenance of long-term depression in rat hippocampal slices in vitro Neurosci Lett 2003 338 147 150 12566174 10.1016/S0304-3940(02)01400-3 Frey U Huang YY Kandel ER Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons Science 1993 260 1661 1664 8389057 Dudek SM Bear MF Bidirectional long-term modification of synaptic effectiveness in the adult and immature hippocampus J Neurosci 1993 13 2910 2918 8331379 Frey U Schollmeier K Reymann KG Seidenbecher T Asymptotic hippocampal long-term potentiation in rats does not preclude additional potentiation at later phases Neuroscience 1995 67 799 807 7675206 10.1016/0306-4522(95)00117-2 Lee HK Kameyama K Huganir RL Bear MF NMDA induces long-term synaptic depression and dephosphorylation of the GluR1 subunit of AMPA receptors in hippocampus Neuron 1998 21 1151 1162 9856470 10.1016/S0896-6273(00)80632-7 Mulkey RM Endo S Shenolikar S Malenka RC Involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression Nature 1994 369 486 488 7515479 10.1038/369486a0 Mulkey RM Herron CE Malenka RC An essential role for protein phosphatases in hippocampal long-term depression Science 1993 261 1051 1055 8394601 Luscher C Nicoll RA Malenka RC Muller D Synaptic plasticity and dynamic modulation of the postsynaptic membrane Nat Neurosci 2000 3 545 550 10816309 10.1038/75714 Abraham WC Williams JM Properties and mechanisms of LTP maintenance Neuroscientist 2003 9 463 474 14678579 10.1177/1073858403259119 Lee HK Barbarosie M Kameyama K Bear MF Huganir RL Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity Nature 2000 405 955 959 10879537 10.1038/35016089 McBain CJ Mayer ML N-methyl-D-aspartic acid receptor structure and function Physiol Rev 1994 74 723 760 8036251 Tovar KR Westbrook GL Mobile NMDA receptors at hippocampal synapses Neuron 2002 34 255 264 11970867 10.1016/S0896-6273(02)00658-X Carroll RC Zukin RS NMDA-receptor trafficking and targeting: implications for synaptic transmission and plasticity Trends Neurosci 2002 25 571 577 12392932 10.1016/S0166-2236(02)02272-5 Snyder EM Philpot BD Huber KM Dong X Fallon JR Bear MF Internalization of ionotropic glutamate receptors in response to mGluR activation Nat Neurosci 2001 4 1079 1085 11687813 10.1038/nn746 Man HY Ju W Ahmadian G Wang YT Intracellular trafficking of AMPA receptors in synaptic plasticity Cell Mol Life Sci 2000 57 1526 1534 11092447 Bredt DS Nicoll RA AMPA receptor trafficking at excitatory synapses Neuron 2003 40 361 379 14556714 10.1016/S0896-6273(03)00640-8 Huang YY Kandel ER Recruitment of long-lasting and protein kinase A-dependent long-term potentiation in the CA1 region of hippocampus requires repeated tetanization Learn Mem 1994 1 74 82 10467587 Williams JM Guevremont D Kennard JT Mason-Parker SE Tate WP Abraham WC Long-term regulation of N-methyl-D-aspartate receptor subunits and associated synaptic proteins following hippocampal synaptic plasticity Neuroscience 2003 118 1003 1013 12732245 10.1016/S0306-4522(03)00028-9 Hessler NA Shirke AM Malinow R The probability of transmitter release at a mammalian central synapse Nature 1993 366 569 572 7902955 10.1038/366569a0 Zilberter Y Uteshev V Sokolova S Khodorov B Desensitization of N-methyl-D-aspartate receptors in neurons dissociated from adult rat hippocampus Mol Pharmacol 1991 40 337 341 1716728 Rosenmund C Feltz A Westbrook GL Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons J Neurophysiol 1995 73 427 430 7714587

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1171533314510.1186/1471-2105-5-117SoftwareXHM: A system for detection of potential cross hybridizations in DNA microarrays Flikka Kristian [email protected] Fekadu [email protected] Astrid [email protected] Inge [email protected] Computational Biology Unit, Bergen Center for Computational Science, UNIFOB/UiB, Thormoehlensgt.55, N-5008 Bergen, Norway2 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NO-7489 Trondheim, Norway3 Sars International Centre for Marine Molecular Biology, Bergen High Technology Centre, Thormoehlensgt. 55, N-5008 Bergen, Norway4 Department of Informatics, University of Bergen, PB. 7800, N-5020 Bergen, Norway2004 27 8 2004 5 117 117 14 5 2004 27 8 2004 Copyright © 2004 Flikka et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results. Results We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at . Conclusions Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results. ==== Body Background The development of DNA microarrays has revolutionized high throughput gene expression analysis. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides [1], and oligonucleotide microarrays, where oligonucleotides in situ synthesized or spotted on glass slides are used [2,3]. In a typical cDNA microarray experiment, total cellular RNA from two sources, a reference (or control) and an experimental sample are converted to cDNA by reverse transcription, labeled with two different fluorescent colors, and hybridized to an array of cDNA probes. One of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array [4,5]. Cross-hybridizations that may arise due to poly(A)-tail of mRNA or repetitive elements may be reduced or eliminated using hybridization blocking reagents like poly(A) oligonucleotides [6] or Cot1 DNA. Another major source of cross-hybridization may come from conserved sequences shared by two or more cDNAs, such as gene family members [4,7]. Analysis of sequenced multicellular eukaryotic genomes suggests that a large percentage of genes belong to gene families, some of which have high sequence similarities [8]. A recent study reported that approximately 17% of human transcripts in the UniGene database contain perfect match repeats of 20 bp minimum lengths [9]. From this study, however, the frequency of long, non-perfect match sequences such as those shared among paralogs, was not clear. Some studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments [3,4,7,9,10]. In general, these studies indicate that cDNAs with nucleotide sequence identities higher than 70–80% over a certain length show significant levels of cross-hybridization. Analysis of chemokines, cytochrome P450 isozymes, G proteins and protease homologous gene families showed that cross-hybridization signals of 0.6–12% and 26–57% could arise from shared nucleotide identities of 55–80% and >80%, respectively [4]. Using synthetic 50 mer oligonucleotides, it was also shown that non-target sequences with >75–80% identity to 50 mer probes in microarrays could result in cross-hybridization [3]. In addition, cross-hybridization was also observed if a non-target sequence included stretches of ≥ 15 continuous bases identical to a 50 mer probe sequence [3]. Cross-hybridization is thought to contribute to discrepancies of results observed between oligonucleotide arrays and cDNA arrays [11]. Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3'- or 5'-ends, the complete sequences of the spotted probes may not be available. We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz [12], OligoWiz [13] and PROBEmer [14], but these are for designing specific probes by avoiding regions of the cDNAs where there may be a cross-hybridization problem. For the assessment of the cross-hybridization potential in DNA microarray analysis, a more specific tool is required. In the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria. The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols – in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization. We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings [3,4], and assess different (transcript) databases in order to evaluate their suitability for our purpose. A web tool is developed that allows the user to query precomputed results from several probe sets, or to analyze own probes with respect to a database representing transcripts in the organism where the microarray experiments are performed. The tool is applicable to analysis of both oligonucleotide and cDNA probes. Below we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse. Implementation The XHM system queries the given nucleotide sequences (for example, a list of differentially regulated genes from DNA microarray experiments) against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria. In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes. Definitions For the calculation of melting temperature, the following formula is used [15]: Tm = 81.5 + 16.6 * log [Na+] + 41 * (numG + numC)/n - 500/n where [Na+] is the Na+concentration, numG and numC is the number of Gs and Cs in the given alignment, and n is the total number of nucleotides aligned. We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays [3]: • Type A similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch). • Type B similarities for identical segments of some minimum length (short perfect match). To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) [16] was used. The default settings of blastn are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions. Databases and sequences In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k Homo sapiens (Hs) and the 14 k Rattus norvegicus (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [17,18] obtained from Research Genetics (Huntsville, AL). The 15 k Mus musculus (Mm) clone set is from NIA [19]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon [20] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows: • RefSeq Version 2 – RefSeq mRNA collection [21]. Number of sequences: 25377/21200/21724 (mouse/rat/human). • UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) [22]. Number of sequences: 87495/50137/118326 (mouse/rat/human). • TIGR gene index [23] – Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human). • BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat). BeGIn is an alternative database we have developed (using tools described in [24]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat). In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study. Overview of the XHM system The basis of the XHM system is a BLAST search (see Figure 1). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for type A or type B matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect. Appropriateness of BLAST Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets (T1...Tn) for a number of probes (P1...Pn). The simulated targets were ensured to satisfy the criteria described in [3] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not Ti was contained in Pi's hitlist. We found that type A matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST's initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server. A web server A web interface has been designed with two entry points for accessing cross-hybridization information (see Figure 1). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified. When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds. Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input. User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for type A hits (defaults are 75% over 50 bp) and minimum length for type B hits (default is 15 bp), based on the findings described in [3], Na+ concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na+ concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments. Output The output from the XHM system consists of different parts (see Figure 2). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B). Results Choice of database Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table 1. Using a 70% identity threshold for the type A similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown). Application to microarray probes As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for type A similarities, and 25 nucleotides for the type B similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence (type A) and 20 nucleotides perfect identity (type B). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends. Results are shown in Table 1. Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no type A hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%. For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more type A hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits. Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database. The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available. The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy. The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%. Discussion A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets [25,26] are widely used in production of microarrays. Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for type A similarities (at least 70% identity), 15–45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays. Although cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases (not all entries represent unique genes). Also one may argue that the 70% identity threshold is somewhat low. Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot. Conclusions We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays. Availability The XHM system is freely available at . Program code for academic use can be supplied upon request to the author. Author's contributions KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript. Acknowledgments We would like to thank Per Winge at Department of Biology, Norwegian University of Science and Technology (NTNU) for useful discussions on DNA hybridization. Ketil Malde at Department of Informatics, University of Bergen is the main developer of the BeGIn databases for Mouse and Rat. Figures and Tables Figure 1 Main components of the XHM system. One or several query sequences (or identifiers) are entered, a BLAST search is performed, the resulting hits are filtered based on length and similarity, and the results are displayed. The dashed arrow symbolizes the possibility to use a sequence id whose hits to the database have been stored. Figure 2 Screenshot of the XHM web output. The screenshot shows an example output from the XHM system. Table 1 Extent of potential cross-hybridizations in DNA microarray probes. Clone set DB Type A (%) Type B (%) CHS(A,B) = 0 = 1 ≥ 2 ≥ 2* = 0 = 1 ≥ 2 Rn 14 RefSeq 63.3 30.7 6.0 16.3 91.3 6.6 1.7 70/200,25 Rn 14 TIGR RGI 5.2 74.6 20.2 21.3 87.7 8.5 3.8 70/200,25 Rn 14 UniGeneUnique 15.9 63.0 21.2 25.2 84.7 11.1 4.2 70/200,25 Rn 14 BeGIn 26.2 62.8 11.1 15.0 82.3 15.4 2.3 70/200,25 Mm 15 RefSeq 34.3 43.2 22.5 34.2 84.5 5.8 9.7 70/200,25 Mm 15 TIGR MGI 47.3 21.5 31.3 59.0 79.5 8.5 12.1 70/200,25 Mm 15 UniGeneUnique 9.9 49.1 40.1 45.0 76.2 11.6 12.3 70/200,25 Mm 15 BeGIn 5.3 57.0 37.8 40.0 78.7 11.5 9.8 70/200,25 Hs 40 RefSeq 13.1 59.5 27.4 31.5 93.3 4.0 2.7 70/200,25 Hs 40 TIGR HGI 1.0 30.8 68.2 68.9 72.5 18.2 9.3 70/200,25 Hs 40 UniGeneUnique 10.9 60.7 28.4 31.8 87.4 7.7 4.9 70/200,25 Mm Oligo RefSeq 32.5 64.4 3.2 4.7 97.7 1.9 0.3 70/69,20 Mm Oligo TIGR MGI 59.6 26.2 14.2 35.1 95.8 3.2 1.0 70/69,20 Mm Oligo UniGeneUnique 9.6 77.7 12.7 14.0 94.8 4.3 0.9 70/69,20 Mm Oligo BeGIn 15.6 77.1 7.3 8.6 95.7 3.6 0.6 70/69,20 Three cDNA probe sets, human (Hs 40), rat (Rn 14) and mouse (Mm 15), and one mouse oligonucleotide set (Mm Oligo) were checked against cDNA databases (DB). In most cases, a single hit shows that the probe is represented in the DB. Two or more hits indicate potential cross-hybridization. If a hit qualifies as a type A hit, it is not considered to be type B, even if it satisfies the criteria. The percentages represent the number of probes having the given number of hits, relative to the total number of probes in the probe set. The column ≥ 2* shows the number of probes with two or more hits, relative to the total number of the probes that were found in the database. Default BLAST settings were used, except for word-size (9), and mismatch penalty (-1). The column CHS(A,B) shows the Cross-Hybridization Settings used for type A and type B search. Total number of probes: Hs 40: 40000, Rn 14:13447, Mm 15:13797 and Mm Oligo:16463. ==== Refs Schena M Shalon D Davis RW Brown PO Quantitative monitoring of gene expression patterns with a complementary DNA microarray Science 1995 270 467 470 7569999 Lockhart DJ Dong H Byrne MC Follettie MT Gallo MV Ghee MS Mittmann M Wang C Kobayashi M Horton H Brown EL Expression monitoring by hybridization to high-density oligonucleotide arrays Nat Biotechnol 1996 14 1675 1680 9634850 10.1038/nbt1296-1675 Kane MD Jatkoe TA Stumpf CR Lu J Thomas JD Madore SJ Assessment of the sensitivity and specificity of oligonucleotide (50 mer) microarrays Nucleic Acids Research 2000 28 4552 4557 11071945 10.1093/nar/28.22.4552 Evertsz EM Au-Young J Ruvolo MV Lim AC AReynolds M Hybridization cross-reactivity within homologous gene families on glass cDNA microarrays Biotechniques 2001 31 1182 1186 passim 11730025 Afshari CA Perspective: Microarray Technology, Seeing More Than Spots Endocrinology 2002 143 1983 1989 12021158 10.1210/en.143.6.1983 Pan SJ Rigney DR Ivy JL Outliers involving the Poly(A) effect among highly-expressed genes in microarrays BMC Genomics 2002 3 35 12479797 10.1186/1471-2164-3-35 Xu W Bak S Decker A Paquette SM Feyereisen R Galbraith DW Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana. Gene 2001 272 61 74 11470511 10.1016/S0378-1119(01)00516-9 Lander ES Linton LM Birren B Nusbaum C Zody MC Baldwin J Devon K Dewar K Doyle M FitzHugh W Funke R Gage D Harris K Heaford A Howland J Kann L Lehoczky J LeVine R McEwan P McKernan K Meldrim J Mesirov JP Miranda C Morris W Naylor J Raymond C Rosetti M Santos R Sheridan A Sougnez C Stange-Thomann N Stojanovic N Subramanian A Wyman D Rogers J Sulston J Ainscough R Beck S Bentley D Burton J Clee C Carter N Coulson A Deadman R Deloukas P Dunham A Dunham I Durbin R French L Grafham D Gregory S Hubbard T Humphray S Hunt A Jones M Lloyd C McMurray A Matthews L Mercer S Milne S Mullikin JC Mungall A Plumb R Ross M Shownkeen R Sims S Waterston RH Wilson RK Hillier LW McPherson JD Marra MA Mardis ER Fulton LA Chinwalla AT Pepin KH Gish WR Chissoe SL Wendl MC Delehaunty KD Miner TL Delehaunty A Kramer JB Cook LL Fulton RS Johnson DL Minx PJ Clifton SW Hawkins T Branscomb E Predki P Richardson P Wenning S Slezak T Doggett N Cheng JF Olsen A Lucas S Elkin C Uberbacher E Frazier M Gibbs RA Muzny DM Scherer SE Bouck JB Sodergren EJ Worley KC Rives CM Gorrell JH Metzker ML Naylor SL Kucherlapati RS Nelson DL Weinstock GM Sakaki Y Fujiyama A Hattori M Yada T Toyoda A Itoh T Kawagoe C Watanabe H Totoki Y Taylor T Weissenbach J Heilig R Saurin W Artiguenave F Brottier P Bruls T Pelletier E Robert C Wincker P Smith DR Doucette-Stamm L Rubenfield M Weinstock K Lee HM Dubois J Rosenthal A Platzer M Nyakatura G Taudien S Rump A Yang H Yu J Wang J Huang G Gu J Hood L Rowen L Madan A Qin S Davis RW Federspiel NA Abola AP Proctor MJ Myers RM Schmutz J Dickson M Grimwood J Cox DR Olson MV Kaul R Raymond C Shimizu N Kawasaki K Minoshima S Evans GA Athanasiou M Schultz R Roe BA Chen F Pan H Ramser J Lehrach H Reinhardt R McCombie WR de la Bastide M Dedhia N Blocker H Hornischer K Nordsiek G Agarwala R Aravind L Bailey JA Bateman A Batzoglou S Birney E Bork P Brown DG Burge CB Cerutti L Chen HC Church D Clamp M Copley RR Doerks T Eddy SR Eichler EE Furey TS Galagan J Gilbert JG Harmon C Hayashizaki Y Haussler D Hermjakob H Hokamp K Jang W Johnson LS Jones TA Kasif S Kaspryzk A Kennedy S Kent WJ Kitts P Koonin EV Korf I Kulp D Lancet D Lowe TM McLysaght A Mikkelsen T Moran JV Mulder N Pollara VJ Ponting CP Schuler G Schultz J Slater G Smit AF Stupka E Szustakowski J Thierry-Mieg D Thierry-Mieg J Wagner L Wallis J Wheeler R Williams A Wolf YI Wolfe KH Yang SP Yeh RF Collins F Guyer MS Peterson J Felsenfeld A Wetterstrand KA Patrinos A Morgan MJ Szustakowki J de Jong P Catanese JJ Osoegawa K Shizuya H Choi S Chen YJ Initial sequencing and analysis of the human genome Nature 2001 409 860 921 11237011 10.1038/35057062 Wren JD Kulkarni A Joslin J Butow RA Garner HR Cross-hybridization on PCR-spotted microarrays IEEE Eng Med Biol Mag 2002 21 71 75 12012609 10.1109/MEMB.2002.1046118 Girke T Todd J Ruuska S White J Benning C Ohlrogge J Microarray Analysis of Developing Arabidopsis Seeds Plant Physiology 2000 124 1570 1581 11115875 10.1104/pp.124.4.1570 Li J Pankratz M Johnson JA Differential gene expression patterns revealed by oligonucleotide ver sus long cDNA arrays Toxicol Sci 2002 69 383 390 12377987 10.1093/toxsci/69.2.383 Nielsen HB Knudsen S Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays Bioinformatics 2002 18 321 322 11847081 10.1093/bioinformatics/18.2.321 Nielsen HB Wernersson R Knudsen S Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays Nucleic Acids Research 2003 31 3491 3496 12824351 10.1093/nar/gkg622 Emrich SJ Lowe M Delcher AL PROBEmer: a webbased software tool for selecting optimal DNA oligos Nucleic Acids Research 2003 31 3746 3750 12824409 10.1093/nar/gkg569 UCMP Oligos Information Altschul SF Gish W Miller W Meyers EW Lipman DJ Basic Local Alignment Search Tool Journal of Molecular Biology 1990 215 403 410 2231712 10.1006/jmbi.1990.9999 Lennon G Auffray C Polymeropoulos M Soares M The I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their expression Genomics 1996 33 151 152 8617505 10.1006/geno.1996.0177 The I.M.A.G.E. Consortium Access to NIA 15 K Mouse cDNA Clone Set QIAGEN Oligo Sets Pruitt KD Maglott DR RefSeq and LocusLink: NCBI gene-centered resources Nucleic Acids Research 2001 29 137 140 11125071 10.1093/nar/29.1.137 Wheeler DL Church DM Edgar R Federhen S Helmberg W Madden TL Pontius JU Schuler GD Schriml LM Sequeira E Suzek TO Tatusova TA Wagner L Database resources of the National Center for Biotechnology Information: update Nucleic Acids Res 2004 32 D35 D40 14681353 10.1093/nar/gkh073 Quackenbush J Liang F Holt I Pertea G Upton J The TIGR gene indices: reconstruction and representation of expressed gene sequences Nucleic Acids Research 2000 28 141 145 10592205 10.1093/nar/28.1.141 Malde K Coward E Jonassen I Fast sequence clustering using a suffix array algorithm Bioinformatics 2003 19 1221 1226 12835265 10.1093/bioinformatics/btg138 Miki R Kadota K Bono H Mizuno Y Tomaru Y Carninci P Itoh M Shibata K Kawai J Konno H Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18 816 full-length enriched mouse cDNA arrays Proc Natl Acad Sci 2001 98 2199 2204 11226216 10.1073/pnas.041605498 Seki M Narusaka M Ishida J Nanjo T Fujita M Oono Y Kamiya A Nakajima M Enju A Sakurai T Satou M Akiyama K Taji T Yamaguchi-Shinozaki K Carninci P Kawai J Hayashizaki Y Shinozaki K Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray Plant J 2002 31 279 292 12164808 10.1046/j.1365-313X.2002.01359.x

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1221534503210.1186/1471-2105-5-122Methodology ArticleAutomatic annotation of protein motif function with Gene Ontology terms Lu Xinghua [email protected] Chengxiang [email protected] Vanathi [email protected] Bruce G [email protected] Dept. of Biostatistics, Bioinformatics and Epidemiology, Medical University of South Carolina, 135 Cannon St. Suite 303, Charleston, SC 29425, USA2 Dept of Computer Science, University of Illinois at Urbana-Champaign, 1304 W. Springfield Avenue, Urbana, IL 61801 USA3 Center for Biomedical Informatics, University of Pittsburgh, 200 Lothrop Street, Suite 8084, Pittsburgh, PA 15213 USA2004 2 9 2004 5 122 122 16 10 2003 2 9 2004 Copyright © 2004 Lu et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Conserved protein sequence motifs are short stretches of amino acid sequence patterns that potentially encode the function of proteins. Several sequence pattern searching algorithms and programs exist foridentifying candidate protein motifs at the whole genome level. However, amuch needed and importanttask is to determine the functions of the newly identified protein motifs. The Gene Ontology (GO) project is an endeavor to annotate the function of genes or protein sequences with terms from a dynamic, controlled vocabulary and these annotations serve well as a knowledge base. Results This paperpresents methods to mine the GO knowledge base and use the association between the GO terms assigned to a sequence and the motifs matched by the same sequence as evidence for predicting the functions of novel protein motifs automatically. The task of assigning GO terms to protein motifsis viewed as both a binary classification and information retrieval problem, where PROSITE motifs are used as samples for mode training and functional prediction. The mutual information of a motif and aGO term association isfound to be a very useful feature. We take advantageof the known motifs to train a logistic regression classifier, which allows us to combine mutual information with other frequency-based features and obtain a probability of correctassociation. The trained logistic regression model has intuitively meaningful and logically plausible parameter values, and performs very well empirically according to our evaluation criteria. Conclusions In this research, different methods for automatic annotation of protein motifs have been investigated. Empirical result demonstrated that the methods have a great potential for detecting and augmenting information about thefunctions of newly discovered candidate protein motifs. Gene Ontologydata miningsupervised learningprotein motiffeature extractionlogistic regression ==== Body Background With the completion of many genome sequencing projects and advances in the methods of automatic discovery of sequence patterns (see Brazma [1] and Brejova et al [2] for reviews), it is now possible to search or discover protein sequence motifs at the genome level. If one regards protein sequences as "sentences" of the biological language with amino acids as the alphabet, then protein motifs can be considered as words or phrases of that language and determining the function of a motif is equivalent to determining the sense of a word. Identifying biological sequence motifs has been a fundamental task of bioinformatics, which has led to the development of several motif (pattern) databases, such as PROSITE, BLOCKS, SMART and Pfam [3-6]. These databases are usually constructed by studying the set of protein sequences that are known to have certain functions and extracting the conserved sequence motifs that are believed to be responsible for their functions. However, the number of motifs that can be extracted in this way is quite limited, and it has been a major challenge to discover new motifs. With the advent of algorithms and programs that can automatically discover sequence motifs from any given set of sequences [1,2,7-9], it is possible to mine a large number of sequences to find novel motifs without necessarily knowing their functions and to compile a dictionary of biological language accordingly. An essential task involved in the compilation of such a dictionary is to determine the function (the meaning) of newly identified protein motifs. Here, we report development of general methods that can be used to predict the function of protein motifs by mining the knowledge in the Gene Ontology. The Gene Ontology™ (GO) project [10] is a concerted effort by the bioinformatics community to develop a controlled vocabulary (GO terms) and to annotate biological sequences with the vocabulary. A biological sequence is described in three different aspects, namely, biological process, cellular component, and molecular function. The standardized annotation with a controlled vocabulary is the main advantage of Gene Ontology, which facilitates both communications among scientists and information management. Both the number of annotated sequences and the number of GO terms associated with individual sequences in the Gene Ontology database are increasing very rapidly. Moreover, natural language processing techniques are also being used to automatically annotate gene products with GO terms [11,12]. Thus, it can be foreseen that the annotations of protein sequences in the Gene Ontology database will become more and more detailed, and have a great potential to be used as an enriched knowledge base of proteins. The basic approach for determining the function of a motif is to study all the sequences that contain the motif (pattern). Intuitively, if all the functional aspects of the sequences matching a motif are known, we should be able to learn which function is most likely encoded by the motif, based on the assumption that every protein function is encoded by an underlying motif. This means that we would need a knowledge base of protein sequences, in which the functions of a sequence are annotated as detailed as possible. In addition, we would also need prediction methods that can work on a given set of protein sequences and their functional descriptions to reliably attribute one of the functions to the motif that matches these sequences. To determine the function of any novel motif, we would first search the protein knowledge base to retrieve all the functional descriptions of the proteins containing the motif, and then use such prediction methods to decide which function is encoded by the motif. In this research, we use the Gene Ontology database as our protein knowledge base and explore statistical methods that can learn to automatically assign biological functions (in the form of GO terms) to a protein motif. Our approach is based on the observation that the Gene Ontology database contains protein sequences and the GO terms associated with the sequences. In addition, the database also contains information of known protein motifs, e.g. the PROSITE patterns that match the sequences. Thus, the protein sequences in the database provide a sample of potential associations of GO term with motifs, among which some are correct (i.e., the GO term definition matches the functional description of the motif) and some are not. This provides us an opportunity to perform supervised learning to identify discriminative features and use these features to predict whether a new association is correct or not. Current Gene Ontology database is implemented with relational database system, which allows one to perform queries like "retrieve all GO terms associated with the sequences that matches a given motif" and vice versa. However, the database usually returns more than one GO terms that may or may not describe the function of the motif in the query. Thus, we need methods to disambiguate which GO term describe the function of the motif (assign a GO term to a motif) and determine how confident we are as the assignment is concerned. We use statistical approaches to learn from known examples and cast disambiguation task into a classification problem. Furthermore, the probability output by the classifier can be used to represent its confidence for the assignment. Recently, Schug et al [13] published their result of automatically associating GO terms with protein domains from two motif databases – ProDom and CDD [14,15]. Their approach is to use protein domains to BLAST [16] search against GO database and assign the molecular functional GO term from the sequence matching the domains with most significant p-value. They found that, in the database they worked with, most sequences only had one functional GO term. Therefore, they could assign the GO term of a sequence to the motif that matched with highest score with fairly good accuracy. However, due to restrictive assumption that each sequence has only one GO term, their approach can not address the potential problem that a sequence matching a motif has multiple associated GO terms, which is common case now, and how to resolve such ambiguity. Results The data set We use the May 2002 release of the Gene Ontology sequence database (available online [17]), which contains 37,331 protein sequences. For each sequence, a set of GO terms assigned to the sequence is identified, and a set of PROSITE patterns that match the same sequence is also retrieved. If both sets are nonempty, all the possible pattern-term combinations formed by the two sets are produced. Table 1 shows an example association of GO terms with PROSITE motifs. The protein MGI|MGI:97380 from the database is assigned seven GO terms and the sequence also matches two PROSITE patterns. Thus, as cross product of two sets, 14 distinct associations are produced. Note that the same pattern-term association may be observed multiple times within the database. A total of 4,135 GO terms, 1,282 PROSITE motifs, and 2,249 distinct PROSITE-GO associations have been obtained from this database. Using the information stored in the Gene Ontology and PROSITE, we manually judged a set of 1,602 cases of distinct PROSITE-GO associations to determine whether the association is correct or not. The PROSITE-GO association set has been judged in two different ways. One way is to label an association as correct if and only if the definition of the GO term and the PROSITE motif match perfectly according to the annotator. Gene Ontology has the structure of a directed acyclic graph (DAG) to reflect the relations among the terms. Most terms (nodes in the graph) have parent, sibling and child terms to reflect the relation of "belonging to" or "subfamily". The second way of judging GO-PROSITE association is to label an association as correct if the GO term and the PROSITE motif are either exact match or the definitions of GO term and PROSITE motif are within one level difference in the tree, i.e., the definition of GO term and the PROSITE motif have either a parent-child relation or a sibling relation according to the GO structure. Thus we have two sets of labeled PROSITE-GO associations, the perfect match set and the relaxed match set (with neighbors). Both sets are further randomly divided into training (1128 distinct associations) and test (474 distinct associations) sets. Since the test sample size is fairly large, the variance of the prediction accuracy can be expected to be small. Thus we have not considered any alternative split of training and test sets. Measuring term-motif associations Intuitively, we may think of the GO terms assigned to a protein as one description of the function of a protein in one language (human understandable) while the motifs contained in the protein sequence as another description of the same function in a different language (biological). We would like to discover the "translation rules" between these two languages. Looking at a large number of annotated sequences, we hope to find which terms tend to co-occur with a given motif pattern. Imagine that, if the sequences that match a motif are all assigned a term T, and none of the sequences that do not match the motif is assigned the term T, then it is very likely that the motif pattern is encoding the function described by term T. Of course, this is only an ideal situation; in reality, we may see that most of, but not all of the proteins matching a motif pattern would be assigned the same pattern, and also some proteins that do not match the motif may also have the same term. Thus, we want to have a quantitative measure of such correlation between GO terms and motif patterns. A commonly used association measure is mutual information (M.I.), which measures the correlation between two discrete random variables X and Y [18]. It basically compares the observed joint distribution p(X = x, Y = y) with the expected joint distribution under the hypothesis that X and Y are independent, which is given by p(X = x)p(Y = y). A larger mutual information indicates a stronger association between X and Y, and I(X;Y) = 0 if and only if X and Y are independent. For our purpose, we regard the assignment of a term T to a sequence and the matching of a sequence with a motif M as two binary random variables. The involved probabilities can then be empirically estimated based on the number of sequences matching motif M (NM), the number of sequences assigned term T (NT), the number of sequences both matching M and assigned T (NT-M), and the total number of sequences in the database. Table 2 shows the top five terms that have the highest mutual information with PROSITE motif PS00109, which is the specific active-site signature of protein tyrosine kinases, along with the related counts. We set out to test whether we can use mutual information as a criterion to assign a GO term to a PROSITE motif. One approach is to use a mutual information cutoff value c to define a simple decision rule: assign term T to motif M, if and only if I(T;M) ≥ c. For a given cutoff c, the precision of term assignment is defined as the ratio of the number of correct assignments to that of the total assignments according to the cutoff c. In Figure 1, we plot the precision at different mutual information cutoff values. It is easy to see that, in general, using a higher (i.e., stricter) cutoff, the precision is higher; indeed, the Pearson correlation coefficient between the precision and the cutoff is 0.837. This suggests that mutual information is indeed a good indicator of the correlation However, a drawback of such an approach is that, given a motif, sometimes, many observed motif-term associations can have mutual information above the cutoff value, making it difficult to decide which pair is correct. While in other cases, the mutual information of the observed motif-term pairs may all be below the cutoff value, but we still would like to predict what terms are most likely to be appropriate for the motif. To address this problem, we can use a different cutoff strategy, and adopt a decision rule that assigns a GO term to a motif based on the ranking of mutual information, which is a common technique used in information retrieval text categorization [19]. More specifically, for each PROSITE motif M in the annotated data set, all observed motif-term associations containing M are retrieved and ranked according to mutual information, then the term that has highest mutual information is assigned to M. Alternatively, if we use this approach to facilitate human annotation, we can relax the rule to include GO terms that have lower ranks, thus allowing multiple potential GO terms to be assigned to a motif, assuming that a human annotator would be able to further decide which is correct. In this method, the key in making a decision is to select a cutoff rank that covers as many correct associations as possible (high sensitivity) while also retrieves as fewer incorrect associations as possible (high specificity). The optimal cutoff can be determined by the desired utility function. Figure 2 shows the Receiver Operating Characteristic (ROC) curve [20] of assigning GO terms to PROSITE motifs in our data set according to the rank of motif-term associations. The two curves are for the two different labeled association sets (i.e., perfect match and relaxed match) respectively. The areas under the two curves are 0.782 and 0.735 respectively, which can be considered as fairly good. We also plot the precision, also referred to as positive predictive value, in panel B. The precision is calculated as the percent of predicted assignments that are truly correct. As shown in panel B, if we assign the GO terms at the top rank for all PROSITE motifs, 50–70% of the cases will be predicted correctly. As we loosen the threshold to include lower ranked terms, we would assign more terms to a motif, and as expected, precision would decline. But even at rank 5, we still have a precision of about 50%. Also shown in Table 2, with respect to the PROSITE pattern of tyrosine kinase (PS00109), most of the top five associated GO terms are related to kinase activity and the term with the highest rank is the most specific. Predicting motif functions using logistic regression While the mutual information measure appears to give reasonable results, there are three motivations for exploring more sophisticated methods. First, the mutual information value is only meaningful when we compare two candidate terms for a given motif pattern; it is hard to interpret the absolute value. While a user can empirically tune the cutoff based on some utility preferences, it would be highly desirable to attach some kind of confidence value or probability of correctness to all the potential candidate motif-term associations. Second, there may be other features that can also help predict the function (term) for a motif. We hope that the additional features may help a classifier to further separate correct motif-term assignment from wrong ones. Third, there exist many motifs with known functions (e.g., those in the PROSITE database), and it is desirable to take advantage of such information to help predict the functions of unknown motifs. This means that we need methods that can learn from such information. In this section, we show that the use of logistic regression can help achieve all three goals. Specifically, we use logistic regression to combine the mutual information with other features, and produce a probability of correct assignment. The motifs with known functions serve as training examples that are needed for estimating the parameters of the regression function. Feature extraction and parameter estimation We now discuss the features to be used in logistic regression, in addition to the mutual information discussed in the previous section. The goal is to identify a set of features that is helpful to determine whether association of any pair of a GO term and a motif is correct or not, without requiring specific information regarding the function of GO term and motif. For a distinct motif-term pair, we collect following frequency-based features: (1) The number of sequences in which the GO term (T) and PROSITE motif (M) co-occur (NT-M). (2) The number of sequences in which T occurs (NT). (3) The number of sequences in which M occurs (NM). (4) The number of distinct GO terms (G) seen associated with M (NG|M). (5) The number of distinct PROSITE patterns (P) seen associated with T (NP|T). In addition, we also consider, as a feature, the similarity of the sequences that support a motif-term pair. Intuitively, if a motif is conserved among a set of diverse sequences, it is more likely that the motif is used as a building block in proteins with different functions. Thus, the average pair-wise sequence similarity of the sequence set can potentially be used as a heuristic feature in the logistic regression classifier. Given a set of sequences, we use a BLAST search engine to perform pair-wise sequence comparisons. We devised a metric AvgS to measure the averaged pair-wise sequence similarity per 100 amino acids (see methods) and use it as an input feature for classifier. To cast the prediction problem as a binary classification problem, we augment our data set of motif-term pairs with a class label variable Y, so that Y = 1 means correct assignment and 0 means incorrect. We represent a motif-term pair by a vector of features X = (X1,..., Xk), where k is the number of features. The seven features/variables used in our experiments are NT-M, NT, NM, NG|M, NP|T, AvgS, and M.I.. Suppose we have observed n motif-term pairs, then we have n samples of (yi, xi), i = 1, 2, ..., n, where, yi is the correctness label and xi is the feature vector for the corresponding motif-term pair. Our goal is to train a classifier which, when given a motif-term pair and feature vector X, would output a label Y with value 1 or 0. Alternatively, we can also consider building a classifier which outputs a probability that Y = 1 instead of a deterministic label. Thus, our task is now precisely a typical supervised learning problem; many supervised learning techniques can potentially be applied. Here, we choose to use logistic regression as our classification model because it has a sound statistical foundation, gives us a probability of correct assignment, and can combine our features naturally without any further transformation. In order to build a model only with the truly discriminative features, it is a common practice to perform feature selection for logistic regression. We use a combined forward and backward feature selection algorithm. Starting from the intercept, we sequentially add features into the model and test if the log-likelihood increases significantly; we keep the current feature if it does. After the forward selection, we sequentially drop features from the model, to see if dropping a feature would significantly reduce the log-likelihood of the model; if it does, we exclude the feature from the model, otherwise continue. When testing the significance, we use the likelihood ratio statistic G, given by 2l(D|β f)/l(D|β -f), where, l(D|β f) and l(D|β -f) are the log-likelihood of the model with feature f and the model without feature f, respectively. Since we add or drop one feature at a time, G follows χ2 distribution with degree of freedom of 1 [21]. We use the p-value of 0.1 as a significant threshold. Figure 3 illustrates the procedure of feature selection. We found that the average pair-wise similarity of supporting sequence set does not contribute to the model significantly and so excluded it; all other variables contribute to the model significantly. The results of parameters estimation are show in the Table 3. Logistic regression classification After fitting the model using the training set, we tested the model on the test set, i.e., we used the model to compute an output p(Yi = 1|Xi) for each test case. Table 4 shows an example of computed conditional probability of correct assignment for the GO terms associated with the protein motif possible the motif "PS00383", which is the "tyrosine specific protein phosphatases signature and profiles". The table 4 lists top 5 GO terms, which are observed to be associated with the motif and ranked according to the conditional probability returned by logistic regression. As the results from the logistic regression are the conditional probability that an association of a GO term with a given motif is correct, we need to decide the cut off threshold for making decision. We calculate the sensitivity and specificity for a different threshold from 0.1 to 0.9 with a step of 0.1 and plotted the ROC curves as shown in Figure 4. The areas under the logistic regression ROC curves are 0.875 and 0.871 for perfect match and relaxed match test set respectively. The precision of the rules is plotted in panel B, where we see that, as the rule becomes more stringent (using a higher threshold), predictions generally become more accurate. We noticed that the precision on the perfect match test set is more variable. This is probably due to the fact that this data set has fewer cases with Y = 1, thus, a small change in the number of cases introduces a large change in percentage. For example, when the threshold is set at 0.9, only three cases are covered by the rule and two of them are correct, thus percent correct drop to 66%. To see whether the additional features are useful, we also performed ROC analysis using different mutual information cutoff threshold on the perfect match test set. The result is shown in Figure 4 panels C and D. We see that using mutual information alone performs almost as well as logistic regression with additional features. However, the area under the curve (0.816) is smaller than that of logistic regression (0.875), indicating that logistic regression does take advantage of other features and has more discriminative power than mutual information alone. The coefficients β1, β2 and β3 for the three features NT-M, NT and NM, which are also involved in the calculation of mutual information, have a very interesting interpretation – they indicate that the roles of these three variables in the logistic regression model actually are to compromise the effect of mutual information! Indeed, according to the formula of the mutual information, a strong correlation corresponds to a high NT-M, low NT, and low NM, but the coefficients shown in Table 3 clearly suggest the opposite. We believe that this actually corrects one drawback of mutual information – over-emphasizing the correlation but ignoring the support or the strength of evidence. For example, if a term is rare, say occurs only once in the data set, then it would have a very high mutual information value (due to an extremely low NT) with respect to any pattern matched by the sequence to which the term is assigned. But, intuitively, one occurrence is very weak evidence, and at least should be regarded as weaker than when we have a term occurring 10 times in total and co-occurring 9 times with the same motif. The key issue here is that mutual information only reflects the correlation between variables, but does not take into account the strength of evidence, therefore, tends to over-favor the situation where there is a perfect correlation but very little evidence. However, the number of sequences in which the co-occurrence happens, which is called the "support" for the association, is also very important. The coefficients for the other two parameters, NG|M and NP|T, are also meaningful. Their negative signs indicate that the more terms a motif co-occurs with or the more motifs a term co-occurs with, the less likely a particular association is correct. This also makes sense intuitively, since all those co-occurring terms can be regarded as "competing" for a candidate description of the motif's function, so the more terms a motif is associated with, the competition is stronger, and thus the chance that any particular term is a correct description of function should be smaller. Thus, the logistic regression model not only performs well in terms of prediction accuracy but also gives meaningful and logically plausible coefficient values. Discussion In this paper, we explore the use of the Gene Ontology knowledge base to predict the functions of protein motifs. We find that the mutual information can be used as an important feature to capture the association between a motif and a GO term. Evaluation indicates that, even used alone, the mutual information could be useful for ranking terms for any given motif. We further use logistic regression to combine mutual information with several other statistical features and to learn a probabilistic classifier from a set of motifs with known functions. Our evaluation shows that, with the addition of new features and with the extra information provided by the motifs with known functions, logistic regression can perform better than using the mutual information alone. This is encouraging, as it shows that we can potentially learn from the motifs with known functions to better predict the functions of unknown motifs. This means that our prediction algorithm can be expected to further improve, as we accumulate more and more known motifs. Although we have so far only tested our methods on the known motifs, which is necessary for the purpose of evaluation, the method is most useful for predicting the functions of new and unknown motifs. For the future work, we can build a motif function prediction system and apply our algorithm to many candidate new motifs e.g., those discovered using TEIRESIAS, SPLASH or other programs. This would further enable us to perform data mining from the Gene Ontology database in several ways. For example, we can hypothesize the functions of a large number of novel motifs probabilistically, then we will be able to answer a query, such as "finding the five patterns that are most likely associated with the GO term tyrosine kinase". This is potentially very useful because it is not uncommon that substantial knowledge about the functions and sub-cellular location of a given protein is available even though a structural explanation for the functions remains obscure. On the other hand, we believe that our methods will facilitate identifying potentially biological meaningful patterns among the millions of patterns returned by pattern searching programs. A sequence pattern that associates with certain GO term with high M.I. or probability is more like to be a meaningful pattern that that with low scores. Furthermore, our methods can also be used in automatic annotation of novel protein sequences as suggested in Schug et al and Rigoutsos et al [9,13,22]. Our methods provide different approaches to associate sequence patterns with functional descriptions. After associating functional descriptions (in the form of GO term) to motifs, we can determine what motifs a novel protein sequence matches and correspondingly transfer the functional descriptions associated with motifs to the sequence. One key advantage of our methods is that the probability of correctness for a GO-motif association can be considered as confidence or uncertainty. This enables one to optimize the automatic annotation according to Bayesian decision theory and minimize the risk of incorrect annotation. Having stated the potential uses of our approaches, we also realize that there exist some limitations for our methods. For example, in order to predict the function of a newly identified sequence pattern correctly, we would require functional annotations of the sequences of GO database be complete and accurate, which may not always be the case. In this paper, we mainly used the motifs with known function to evaluate the capability of the methods developed in this research. Our result shows that the methods work well with known sequences patterns. Currently, the annotation of motif function with GO term is carried out manually at the European Bioinformatics Institute (the GOA project). Such approach is warranted because human annotation is more accurate than automatic ones. However, as the amount of information regarding protein functions accumulates and a large number of new potential motifs are discovered, it will be very labor intensive to annotate the potential association of protein function and protein patterns. By then, the methods studied in this research will potentially prove to be useful to discover the underlying protein motifs that are responsible for the newly annotated function. For example, the methods can be used as prescreening to narrow down to the most possible associations of protein function and motifs, thus facilitate human annotation. Conclusions In summary, we have developed methods that disambiguate the associations between of Gene Ontology terms and protein motifs. These methods can be used to mine the knowledge contained in the Gene Ontology database to predict the function of novel motifs, discover the basis of a molecular function at primary sequence level and automatically annotated the function of novel proteins. Methods Mutual information Mutual information is defined as follows In which the probabilities p(X = x, Y = y), p(X = x) and p(Y = y) can be empirically estimated from the data by counting occurrence/co-occurrence followed by normalization. Sensitivity and specificity The sensitivity and specificity of the rules are calculated as where TP (True Positive) is the number of associations labeled as correct among the retrieved motif-term pairs meeting the ranking cutoff criteria, FN (False Negative) is the number of associations labeled as correct but not retrieved, TN (True Negative) is the number of associations labeled as incorrect and not retrieved, and FP (False Positive) is the number of associations labeled incorrect but are retrieved. Averaged sequence similarity Calculation of the average pair-wise sequence similarity per 100 amino acids (AvgS) of a sequence set is as follows Where Sij is raw BLAST pair-wise similarity scores between the sequence i and sequence j; Li and Lj are the lengths of sequences i and j, respectively; n is the number of sequences in the set; and δ (i, j) is a delta function which equals 1 if i = j and 0 otherwise. Logistic regression The logistic regression model is a conditional model that assumes the following linear relationship between p(Y = 1|X) and X1, ..., Xk: where, β = (β0, β1, ..., βk) is the parameter vector. We can fit the logistic regression model (i.e., estimate the parameters) using the Maximum Likelihood method – essentially setting the parameters to values at which the likelihood of the observed data is maximized (Hosmer and Lemeshow 1989, Hastie et al 2001). In our experiments, we use iteratively reweighted least squares (IRLS) algorithm [23] to fit the logistic regression model. All features are normalized to zero mean and unit variance before training. Acknowledgments This research is partially supported by National Library of Medicine (NLM) training grant to Lu, X (No. 3 T15 LM07059-15S1), Gopalakrishnan, V (No. 5 T15 LM07059-15) and NLM grant to Buchanan, B.G. (No. LM06759). We would like to thank Drs. Roger Day, Milos Hauskrecht and Gregory Cooper for insightful discussions. Figures and Tables Figure 1 Correlation of mutual information cutoff and term assignment precision. Different M.I. cutoff value is used to assign GO terms to motifs. The precision of assignment is plotted vs M.I. cutoff value. The Pearson correlation coefficient between the precision and the cutoff is 0.837. Figure 2 Assigning GO term to motif according to rank of M.I. A. ROC curves of assigning GO terms to motifs according to rank of mutual information. The filled circle is for the perfect match data set, and the area under the curve is 0.782. The empty triangle is for the relaxed match data set, and the area under the curve is 0.735. The numbers next to data points indicate cut-off ranks of decision rules. Diagonal line corresponds to random model. B. Precision of rules based on different mutual information cutoff ranks. Filled bars are results on the perfect match data set. Empty bars are the results on the relaxed match data set. Figure 3 The algorithm for feature selection. Figure 4 Comparison of results with probability and M.I. A. ROC curves for classifying motif-term associations at different probability threshold. Filled circles are the results on the perfect match test set with an area under curve of 0.8715. Empty triangles are on the relaxed match test set with an area under curve of 0.871. Data points correspond to thresholds of p(Y = 1|X) from 0.9 to 0.1 (from left to right) with a step of 0.1. B. Precision (positive predictive value) at different probability cutoffs, where solid bars are the result on the perfect match test set and the open bars for the relaxed match test set. C. ROC curve for decision rules based on different M.I. cutoff thresholds with an area under curve of 0.816. D. Precision at different M.I. cutoffs. Table 1 GO terms and PROSITE patterns for the protein MGI|MGI:97380 GO Terms and Descriptions PROSITE Motifs and Descriptions GO:0005576 Extracellular PS00248 Nerve growth factor family signature and profile (1) GO:0005515: Protein-binding PS50270 Nerve growth factor family signature and profile (1) GO:0005166: neurotrophin p75 receptor ligand GO:0008544 epidermal differentiation GO:0007422 peripheral nervous system development GO:0007420 brain development GO:0007403 determination of glial fate Table 2 Five GO terms associated with PROSITE pattern PS00109 (tyrosine kinase signature) GO Term GO Definition NM NT NT-M M.I. GO:0004713 Protein tyrosine kinase 246 68 51 0.00599 GO:0006468 Protein amino acid phosphorylation 246 409 69 0.00464 GO:0004714 Transmembrane receptor protein kinase 246 33 29 0.00362 GO:0004715 Non-transmembrane protein tyrosine kinase 246 17 14 0.00168 GO:0005887 Integral membrane protein 246 1162 44 0.00118 Table 3 Result of logistic regression parameters estimation Estimated Coefficients Features On the perfect match set On the relaxed match set Intercept (β0) -1.7549 -0.6263 NT-M(β1) -0.3845 -0.4546 T (β2) 1.3652 1.6827 NM (β3) 1.0497 0.4735 NG|M (β4) -1.9792 -1.1113 NP|T (β5) -1.7883 -2.5494 M.I. (β6) 1.0002 1.1598 Table 4 Top 5 GO terms associated with the motif PS000383 ranked according the conditional probability of correctness of association. The column 2~7 consist of the feature vector for motif-GO association is listed, the conditional probability p(Y = 1|X) is calculated with trained model and the true classes are list in right two columns of the table. The definition of the GO terms is listed at the bottom of the table. GO Terms Input Features (X) p(Y = 1|X) True Class NT-M NT NM NG|M NP|T M.I GO:0006470 65 122 191 95 27 0.007268 0.97577 1 GO:0005001 23 24 191 95 5 0.003177 0.87681 1 GO:0004726 10 11 191 95 10 0.001331 0.68682 1 GO:0005634 11 1785 191 95 281 5.08E-06 0.27882 0 GO:0005887 17 1162 191 95 225 0.000193 0.15536 0 GO:0006470: Protein amino acid dephosphorylation; Cellular Processes GO:0005001: transmembrane receptor protein tyrosine phosphatase activity: Molecular Function GO:0004726: non-membrane spanning protein tyrosine phosphatase activity: Molecular Function GO:0005634: Nucleus; Cellular Component GO:0005887: Integral to plasma memberane; Cellular Component ==== Refs Brazma A Jonassen IDG Approaches to the automatic discovery of patterns in biosequences J Comput Biol 1998 5 279 9672833 Brejova B DiMarco C Vinar T Hidalgo SR Holguin G Patten D Finding Patterns in Biological Sequences Technical Report CS-2000-22, University of Waterloo 2000 Falquet L Pagni M Bucher P Hulo N Sigrist CJ Hofmann K Bairoch A The PROSITE database, its status in 2002 Nucleic Acids Res 2002 30 235 238 11752303 10.1093/nar/30.1.235 Henikoff JG Greene EA Pietrokovski S Henikoff S Increased coverage of protein families with the blocks database servers Nucl Acids Res 2000 28 228 230 10592233 10.1093/nar/28.1.228 Bateman A Birney E Cerruti L Durbin RLE Eddy SR Griffiths-Jones S Howe KL Marshall MELS The Pfam protein families database Nucleic Acids Res 2002 30 276 280 11752314 10.1093/nar/30.1.276 Schultz J Milpetz F Bork P Ponting CP SMART, a simple modular architecture research tool: identification of signaling domains Proc Natl Acad Sci USA 1998 95 5857 5864 9600884 10.1073/pnas.95.11.5857 Lawrence CE Altschul SF Boguski MS Liu JS Neuwald AF Wootton JC Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment Science 1993 262 208 214 8211139 Califano A SPLASH: structural pattern localization analysis by sequential histograms Bioinformatics 2000 16 341 357 10869032 10.1093/bioinformatics/16.4.341 Rigoutsos I Floratos A Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm Bioinformatics 1998 14 229 Consortium TGO Creating the gene ontology resource: design and implementation Genome Res 2001 1425 1433 11483584 10.1101/gr.180801 Raychaudhuri S Chang JT Sutphin PD Altman RB Associating genes with gene ontology codes using a maximum entropy analysis of biomedical literature Genome Res 2002 12 203 214 11779846 10.1101/gr.199701 Xie H Wasserman A Levine Z Novik A Grebinskiy V Shoshan A Mintz L Large-scale protein annotation through gene ontology Genome Res 2002 12 785 794 11997345 10.1101/gr.86902 Schug J Diskin S Mazzarelli J Brunk BP Stoeckert CJJ Predicting gene ontology functions from ProDom and CDD protein domains Genome Res 2002 12 648 655 11932249 10.1101/gr.222902 Marchler-Bauer A Anderson J DeWeese-Scott C Fedorova N Geer LHeS Hurwitz D Jackson J Jacobs A Lanczycki C CDD: a curated Entrez database of conserved domain alignments Nucleic Acids Res 2003 31 383 387 12520028 10.1093/nar/gkg087 Corpet F Servant F Gouzy J Kahn D ProDom and ProDom-CG: tools for protein domain analysis and whole genome comparisons Nucleic Acids Res 2000 28 267 269 10592243 10.1093/nar/28.1.267 Altschul SF Gish W Miller W Myers EW Lipman DJ Basic local alignment search tool J Mol Biol 1990 215 403 410 2231712 10.1006/jmbi.1990.9999 Gene Ontology Consortium download site Cover T Thomas J Elements of information Theory 1991 John Wiley & Sons, Inc. Yang Y An Evaluation of Statistical Approaches to Text Categorization J of Information Retrieval 1999 1 Bradley AP The use of the area under the ROC curve in the evaluation of machine learning algorithms Pattern Recognition 1997 30 1145 1159 10.1016/S0031-3203(96)00142-2 Hosmer DWJ Lemeshow S Applied logistic regression 1989 John Wiley & Sons, Inc. Rigoutsos I Huynh T Floratos A Parida L Platt D Dictionary-driven protein annotation Nucleic Acids Res 2002 30 3901 3916 12202776 10.1093/nar/gkf464 Hastie T Tibshirani R Friedman J The elements of statistical learning Springer 2001

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==== Front BMC Cell BiolBMC Cell Biology1471-2121BioMed Central London 1471-2121-5-321533314410.1186/1471-2121-5-32Research ArticleMutations in the IGF-II pathway that confer resistance to lytic reovirus infection Sheng Jinsong [email protected] Edward L [email protected] Chuanming [email protected] K Sam [email protected] H Earl [email protected] Donald H [email protected] Research Medicine, Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN, 37212, USA2 Department of Medicine, Division of Infectious Diseases, Vanderbilt University, Nashville, TN, USA3 Department of Microbiology and Immunology, Vanderbilt University, Nashville, TN, 37232, USA4 Department of Medicine, Division of Nephrology, Vanderbilt University, Nashville, TN, 37232, USA5 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, 37232, USA2004 27 8 2004 5 32 32 21 7 2004 27 8 2004 Copyright © 2004 Sheng et al; licensee BioMed Central Ltd.2004Sheng et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Viruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies. Results A gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo. Conclusion These results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection. ==== Body Background Viruses as obligate intracellular parasites rely upon the host cell for different steps in their life cycle, including attachment, disassembly, transcription, translation, reassembly, and egress. Consequently, characterization of these cellular processes will be essential for any understanding of viral life cycles, and may provide cellular targets for new antiviral therapies. The susceptibility to virus infection varies greatly among different cell types, and virus-resistant cells frequently emerge post-infection [1-4]. This suggests that host cell contributions to the virus life cycle, although complex, have genetic determinants. We therefore used a genetic approach to identify cellular genes required for infection by reovirus, a small cytolytic RNA virus that replicates in the cytoplasm. A gene trap retrovirus was use to create libraries of rat intestinal epithelial (RIE-1) cell clones in which each clone contained a single gene disrupted by an integrated retrovirus. The mutant libraries were then infected with reovirus, and resistant clones were selected. We hypothesized that genes mutated by gene entrapment may confer reovirus resistance as a result of either haploinsufficiency or loss of heterozygosity and could be identified by characterizing the genes disrupted by the entrapment vector. From these experiments we have isolated 152 clones and have characterized mutations in 111 different genes, providing potential candidates required for reovirus infection and/or cell killing. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, four mutations affected genes in the insulin growth factor-2 (IGF-II) pathway, including genes encoding the IGF-ll/manose-6-phosphate receptor [5,6] (Igf2r, locus ID 25151), the IGF binding protein 5 protease [7] (Prss11, locus ID 65164, 2 clones), and CTCF (Ctcf, locus ID 83726), a transcriptional repressor of the IGF-II gene (Igf2) involved in paternal imprinting. The frequency of mutations involving the IGF-II pathway led us to investigate the role of IGF-II in reovirus infection. Clone 6B72, which contains a mutation in Ctcf [8], was found to over express Igf2 transcripts, consistent with the known role of CTCF as a transcriptional repressor of the Igf2 gene. Moreover, forced expression of IGF-II in RIE-1 cells was sufficient to confer cellular resistance to lytic reovirus infection. Enforced IGF-II expression also transformed RIE-1 cells to anchorage independent growth, a phenotype associated with malignant change. These results represent the first use of gene entrapment to identify components of host cell metabolism required for virus infection and illustrate a potential mechanism of carcinogenesis associated with chronic virus infection. Results Disruption of the CTCF gene results in cells resistant to lytic infection by reovirus Gene entrapment strategies to identify host genes required virus replication depend on methods to select for virus resistant clones present at about one in 104–105 mutagenized cells. Unfortunately, cells persistently infected with reovirus (PI) can emerge at high frequencies (one in 102–103) and are intrinsically resistant to the virus [9,10]. In preliminary studies, we found that hepa 1/a cells were not suitable for genetic studies due to the emergence of PI clones. However, persistently infected rat intestinal epithelial cells (RIE-1) [11,12] were found to require a serum survival factor and die when placed in serum free media (Figure 1). In vitro infection of RIE-1 cells with reovirus also appears to mirror virus replication in the rodent host [13-15]. Consequently, RIE-1 cells were used in the present study, and reovirus-resistant clones were selected in serum-free medium to remove PI survivors. RIE-1 cells were mutagenized by using the U3NeoSV1 gene trap shuttle vector [16] (see Methods) and the resulting libraries of mutagenized cells were infected with reovirus serotype 1/Lang at a multiplicity of infection (MOI) of 35, to select for clones resistant to lytic infection. The isolated clones did not express reoviral antigens, and did not produce infectious virus as assessed by plaque assay, suggesting these reovirus resistant clones were not PI. Figure 1 Persistently infected RIE-1 cells fail to survive in serum-free media. RIE-1 parental cells and cells persistently infected with reovirus type 1 were plated in complete medium (FBS+) or in media in which the serum was omitted (FBS-). Surviving cells were stained with gentian violet after 7 days. Darkly staining wells represent cell survival. Regions of genomic DNA adjacent to the U3NeoSV1 provirus in each virus resistant clone were isolated by plasmid rescue and sequenced. Altogether, of the 151 isolated clones, 62% of flanking sequences matched known or presumptive genes, and an additional 23% were represented in the public databases of expressed sequence tags (dbEST) or non-redundant sequences (nr). From the 111 clones matching known or presumptive genes, 10 genes were represented more than once. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, the library included 4 independent mutations involving three genes that encoded proteins associated with the insulin growth factor-2 (IGF-II) signalling pathway, namely, IGF-ll/manose-6-phosphate receptor [5,6] (Igf2r, locus ID 25151), the IGF binding protein 5 protease [7] (Prss11, locus ID 65164, 2 clones), and CTCF (Ctcf, locus ID 83726), a transcriptional repressor of IGF-II involved in paternal imprinting [17,18]. The position of the provirus (clone 6B72) in the first intron of the Ctcf gene is shown in Figure 2. CTCF differentially represses maternal Igf2 gene expression, whereas the imprinted paternal gene escapes repression due to methylation of CTCF binding sites [17,18]. Figure 2 Disruption of the Ctcf gene in 6B72 cells. DNA sequences flanking the U3NeoSV1 provirus in 6B72 cells were cloned and sequenced. The flanking sequences were identical to sequences in the rat genome, placing the provirus in the first intron of the Ctcf gene (A). Filled and open boxes indicate coding and non-coding exons, respectively. Flanking sequences 5' of the U3NeoSV1 provirus (B) include the first exon (shaded) of the gene. Igf2 transcripts induced by CTCF disruption Considering that four clones had mutations in the IGF-II pathway, the relationship between the Ctcf mutation in 6B72 cells and the virus resistant phenotype was investigated further. Levels of CTCF protein in 6B72 cells were reduced by about 50% as assessed by western blot analysis (Figure 3A), consistent with the disruption of one allele. Diminished CTCF expression was associated with an increase in Igf2 transcripts as assessed by Northern blot hybridization (Figure 3B). In addition, two products were amplified from 6B72 cells by RT-PCR using primers flanking the IGF-II pro-hormone coding sequence (Figure 3C). The first product was identical to the rat IGF-II pro-hormone (pro-IGF-II) coding sequence (Accession X17012), whereas, the second contained 14 additional nucleotides generated by splicing of exon 2 to an alternative splice acceptor located 14 nucleotides upstream of the normal Igf2 exon 3 splice acceptor site. The alternative transcript is expected to encode a pro-IGF-II protein extending 11 amino acids into the E-peptide sequence ending in a stretch of 60 amino acids lacking homology with any known protein (Figure 4). Figure 3 CTCF and Igf2 expression in RIE-1 and 6B72 cells. Levels of CTCF protein were assessed by Western blot analysis (A), normalized to a β-actin control. Levels of Igf2 transcripts were assessed by Northern blot analysis (B) normalized to GAPDH control. Protein content, as assayed by western blot analysis and standardized to β-actin was decreased in the 6B72 cell clone to 30% of control. Reverse transcriptase PCR analysis of Igf2 transcripts (C). The RT-PCR products (arrows) were separated on a 2% agarose gel, revealing an additional transcript in the 6B72 cells. The DNA sequence of the larger RT-PCR product (D) revealed an alternatively spliced transcript (Igf2sv) generated by splicing of exon 2 to a cryptic 3' splice site located 14 nucleotides upstream of exon 3. Figure 4 Alternatively spliced product contains a single nucleotide polymorphism in the igf2 coding sequence. Intron sequences incorporated into the alternatively spliced transcript (highlighted in black) alter the translational reading frame of the pro-homone downstream of the coding sequence of the processed IGF-II protein (italics and bold). The Igf2sv PCR product also contained a G to A base substitution (underlined) that replaces alanine with threonine at codon 62 (boxed) of the mature hormone. Resistance to reovirus lytic infection results from increased Igf2 expression The stability of virus resistance in the 6B72 cell was tested by infecting RIE-1 and 6B72 cells with reovirus type 1 at a MOI of 10. There was approximately a 10 fold lower titre of reovirus obtained following infection of 6B72 cells as compared to RIE-1 cells at 24 hrs. post-infection (4.5 × 105 versus 5.1 × 106), and the difference was also maintained at 48 hours post-infection (data not shown). Additionally, there was a dramatic difference in the survival of 6B72 cells after being exposed to high titres of reovirus type 1 (Figure 5). Figure 5 IGF-II modulates reovirus resistance in RIE-1 and 6B72 cells. RIE-1 and 6B72 cells expressing either the IGF-II pro-hormone (proIGF2) or the alternatively spliced transcript (IGF2SV, Figure 3D) were challenged with serial dilutions of reovirus type 1 (upper panel), and the surviving cells were stained with gentian violet 4 days post-infection. The multiplicity of infection (MOI) for each row is indicated. pro-IGF-II converted RIE-I cells to a reovirus resistant phenotype (RIE-1/proIGF2) but had little if any effect on already-resistant 6B72 cells (6B72/proIGF2). Plasmids expressing the alternatively spliced Igf2 transcript had no effect on RIE-1 cells (RIE-1/IGF2SV) but abrogated virus resistance in 6B72 cells (6B72/IGF2SV). The experiment was repeated 3 times. Expression of Igf2 transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. Expression of Igf2 in 6B72 is not shown. To determine whether Igf2 confers resistance to reovirus infections, clones of RIE-1 cells over-expressing the full-length Igf2 transcript or the splice variant (Igf2sv) were generated and examined for their capacity to resist lytic infection. As shown in Figure 5 expression of wild type (Igf2), but not the splice variant (Igf2sv) increased the resistance of RIE-1 cells to reovirus infection by over 100 fold. However, when the Igf2svwas transfected into 6B72 cells, the ability of 6B72 cells to survive infection was abolished. Expression of the Igf2 gene in an anti-sense orientation caused no significant difference in the capacity of 6B72 cells to resist infection (data not shown). These studies suggest that increased Igf2 expression in 6B72 cells is associated with their capacity to resist reovirus infection, and that the Igf2svencodes a trans-dominant isoform that blocks the activity of Igf2. The IGF-II hormone confers resistance to lytic infection Igf2 transcripts encode a pro-hormone of 180 amino acids that is processed to generate the 67-residue IGF-II protein [19]. Other proteolytic products including the 89-residue carboxy-terminal E peptide may also be biologically active [20]. The Igf2sv PCR product also contained a single nucleotide substitution (G1393A) that results in the substitution of a threonine for alanine at position 62 of IGF-II, raising questions about Igf2 sequences that influence reovirus resistance. Vectors expressing only the 68-residue IGF-II protein both with and without the A62T change were compared for their ability to confer resistance to lytic infection by reovirus type 1 (Figure 6). The IGF-II expression vector converted RIE-1 cells to a virus resistant phenotype; whereas, the IGF-II62T expression plasmid was inactive (Figure 6) and did not suppress the resistance of 6B72 cells (data not shown). These results indicate that virus resistance can be affected by mutations in the IGF-II coding sequence and downstream sequences, including the E-peptide, are not required. However, the trans-dominant effects of Igf2sv apparently require alterations to the carboxy-terminus of the IGF-II pro-hormone. Figure 6 IGF-II sequences lacking the E-peptide can convert RIE-1 cells to a reovirus-resistant phenotype. Two independent clones of RIE-1 cells transfected with plasmids expressing native (IGF-II) and mutant (IGF-IIA62T) proteins without the carboxyl terminal extension-peptide (E-peptide) were challenged with serial dilutions of reovirus type 1 as described in Figure 4. Native IGF-II protected RIE-1 cells from reovirus infection (IGF-II(a) and IGF-II(b)), while the IGF-IIA62T mutant (IGF-IIA62T(a) and IGF-IIA62T(b) did not. Non-infected RIE-1 cells (C), and infected 6B72, and RIE-1 cells were included as controls. Expression of Igf2 transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. The native IGF-II (small arrow) is slightly larger than the cDNA constructs (larger arrow), whereas the double-sided arrow marks the constitutively expressed GAPDH, as shown. Expression of Igf2 in 6B72 is not shown. Over expression of igf2 confers resistance to other reovirus sertotypes RIE-1 cells are intrinsically resistant to infection by reovirus type 3 [13]; therefore, experiments to assess the effects of IGF-II expression on lytic infection by reovirus type 3, were performed using murine L-cells. As shown in Figure 7, L-cells expressing Igf2 were significantly more resistant to both reovirus serotypes than the parental cells, indicating that the ability of IGF-II to confer resistance to reovirus infection is not limited to a single cell or virus type. Figure 7 Decreased lytic infection of L-cell clones over expressing the IGF-II gene. Constitutively expressed GAPDH was used to assess loading of RNA in lanes. Survival of lytic infection was determined by infecting 105 L-cells or L-cells expressing the pro-IGF2 transgene with varying concentrations of reovirus type 1 (A) or type 3 (B). The multiplicity of infectious virus particles per cell (MOI) is indicated for each virus serotype. Surviving cells were visualized at 4 (A) or 5 days (B) following infection with gentian violet. Transgene expression was determined by northern blot (C). Experiments were repeated three times and a representative experiment is shown. IGF-II expressing RIE-1 cell has delayed disassembly of virus particle Previous studies have indicated that proteoytic disassembly of virus particles occurs in the lysosome and requires the participation of cathepsin B and L that are transported by the Igf2r gene [21-23]. Igf2 binds to Igf2r resulting in alteration in cathepsin trafficking [24]. Georgi and colleagues showed that directly fluorescentated reovirus particles dissipated their fluorescent signal coincident with disassembly of the outer capsid [25]. To determine whether over expression of igf2 would affect disassembly of virions, we examined the fate of virus during the first hours of infection. Purified reovirus type 1 virions were directly labeled with fluorescein and adsorbed to RIE-1 or 6B72 cells, and the persistence of a fluorescein signal determined at 2 hrs following virus absorption (fig 8A,8B). The binding of virions to RIE-1 cells and 6B72 cells and initial accumulation of virus particles within cells was similar, however fluorescence was almost non-detectable in RIE-1 cells (Figure 8A) whereas it was still present in 6B72 cells at 2 hrs (Figure 8B). In addition, western blot analysis of virion proteins 2 hrs following attachment to cells confirmed the persistence of the σ3 protein in the 6B72 cell clone, but not RIE-1 cells (fig 8C,8D). Therefore, disassembly of virions is altered in the 6B72 cell clone. Figure 8 6B72 cells have delayed disassembly of reovirus type 1. Fluorescein-labelled reovirus particles were absorbed to RIE-1 (A) or 6B72 cells (B). Persistent fluorescence at 2 hours was found in 6B72 cells, but not in RIE-1 cells. Non-replicating reovirus type 1, at 3 × 104 particles per cell, was adsorbed to RIE-1 (C) or 6B72 (D) cells at 4°C, washed and incubated at 37°C for 2 and 4 hours. Cells were lysed and the state of virus particles determined by western blot. The outer capsid proteins μ1 and σ3 are present in the 6B72 cell preparations at 2 and 4 hours, but not in the RIE-1 cells. CTCF deficient RIE cells grow in soft agar, a consequence of increased Igf2 expression Accumulating evidence suggests that enhanced IGF-II expression associated with loss of Igf2 genomic imprinting may promote tumour formation [26,27]. Although RIE-1 and 6B72 cells appeared to proliferate at similar rates as assessed by MTS/PMS incorporation (Data not shown), 6B72 cells that have a disrupted Ctcf gene were capable of forming colonies in soft agar (Figure 9b). To assess the role of IGF-II in anchorage-independence, cells transfected with Igf2 and Igf2sv were tested for their ability to grow in soft agar. RIE-1 cells transfected with Igf2, but not Igf2sv formed colonies in soft agar (Figure 9a), whereas 6B72 cells transfected with Igf2sv lost their capacity to proliferate in soft agar as did cells transfected with an anti-sense Igf2 construct (Figure 9b). The IGF-II62T expression plasmid had no transforming activity (Figure 9d). On balance, transforming activities of the different Igf2 plasmids on RIE-1 and 6B72 cells as assessed by the soft agar assay were similar to their effects on reovirus resistance, although the vector expressing only IGF-II was less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone. Figure 9 Anchorage-independent growth phenotypes of RIE-1 cell clones. 105 cells were suspended in media containing 1% agarose and plated in 6 well culture dishes. RIE-1 cells acquire the ability to grow in soft agar after being transfected with a vector expressing pro-IGF2 but not the IGF2SV splice-variant (a). The vector inserted in the Ctcf gene (6B72) confers the ability to grow in soft agar, but the phenotype is suppressed by expression of IGF2SV (b). Clones selected for reovirus resistance with gene trap vectors inserted into the Prss11, Igf2r and Anxa2 genes failed to grow in soft agar (c). RIE-1 cells expressing native IGF-II protein without the E-peptide grew in soft agar but the colonies were smaller (d) than produced by pro-IGF-2 (a), while the corresponding IGF-IIA62T protein (E-peptide) did not transform RIE-1 cells to anchorage independence (d). Colonies were photographed (20×) after 7 days except (d) where the cells were photographed after 10 days. The capacity to proliferate in soft agar was not a property of other reovirus resistant cell clones that contained mutations in the IGF-II pathway. Figure 9 shows that cell clones with disrupted Igfr or Prss11 genes did not form colonies in soft agar (Figure 9c). An additional reovirus resistant-clone with an insert in the Anxa2 (annexin 2) gene, associated with cytomegalovirus infection [28] and recognized to bind to the insulin and insulin growth factor receptor-1 [29], also failed to grow in soft agar (Figure 9c). Therefore, the capacity to proliferate in soft agar was not a general property of reovirus-resistant cells, even in clones that contain mutations in the IGF-II pathway. However, L-cells also displayed enhanced ability to grow in soft agar in addition to virus resistance, following enforced Igf2 expression (Figure 10). Figure 10 IGF-II increases colony formation of L-cells in soft agar. Forced expression of the rat pro-Igf2 gene (pro-Igf2c) in L-cells increases the number of soft-agar colonies by 4 to 5 fold as compared to the L-cell parent, as shown in this representative photomicrograph at 7 days (10×). Discussion Insertional mutagenesis provides an approach to identify genes associated with selectable cellular phenotypes. We have isolated over 100 potential clones with mutations in genes that may play roles in the life cycle of reovirus. In the present study, one clone resistant to reovirus lytic infection contained a provirus inserted into the gene encoding the CTCF transcriptional regulator. CTCF binding motifs are present in many genes, including Igf2 [26], H19 [27], and Myc [8]. However, since 3 other clones selected for reovirus resistance contained mutations in the IGF-II pathway, the role of IGF-II in virus-resistance was investigated further. Reduced expression of the Ctcf gene was associated with enhanced Igf2 expression in virus-resistant cells, while forced expression of the Igf2 gene in the parental RIE-1 line was sufficient to confer resistance to lytic reovirus infection. By inference, the recovery of inserts affecting other genes in the IGF-II signalling pathway suggests that mutations in multiple genes may affect the same phenotype by acting on a common pathway. The insert in Igf2r was found to decrease the expression of the gene as assayed by northern blot analysis (data not shown). As IGF-II targets the igf2r to lysosomal degradation, mutations in the genes encoding either the receptor (Igf2r) or its ligand (Igf2) will affect the activity of the other, and result in a reduced endosomal trafficking of hydrolases necessary for reovirus disassembly [24]. Our data indicates there is a decrease in virus disassembly in 6B72 cells, consistent with a block at this step in morphogenesis. Further studies will be required to assess if and how inserts in the Prss11 and Igf2r genes influence reovirus resistance. As the entry, disassembly, transcription, translation, and repacking of viruses share common features; we anticipate that common cellular pathways will influence infection by other virus families. Indeed, Igf2r has been implicated in herpes simplex and zoster virus infection [30,31]. However, the present study is the first to show a direct connection between Igf2 gene expression and resistance to lytic virus infection. By using constructs that encode the mature hormone without the E-peptide, we were able to show that forced IGF-II expression is sufficient to confer a reovirus resistant phenotype. These results differ from other studies in which reovirus replication was enhanced by treatment of RIE-1 cells with insulin. The latter effect is presumably caused by enhanced virus replication associated with cell proliferation [13]. Expression of the Igf2 gene is frequently elevated in common childhood and adult neoplasms [27,32-38] and has been associated with tumour progression and metastasis [39,40]. Igf2 also transformed RIE-1 cells to anchorage-independence, a phenotype that predicts the potential for malignant growth in vivo. Virus-resistant 6B72 cells also grew in soft agar, presumably as a result of enhanced IGF-II expression. Since plasmids expressing only IGF-II were less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone, further study is needed to determine whether the capacity of RIE-1 cells to proliferate in soft agar is enhanced by the E-peptide or other products derived from the carboxy terminus of the pro-hormone. In other studies, the E-peptide enhanced insulin secretion from β-cells [20], and may play a role in cellular transformation [31]. cDNA clones of an alternatively spliced Igf2 transcript (Igf2sv), blocked the ability of IGF-II to promote reovirus resistance and anchorage independent growth in a trans-dominant manner. The protein coding sequence of Igf2svcontains a frame shift in the E-peptide region and lacks a site [20,41] required for the proteolytic processing of the pro-hormone. The alternative splice site is used very infrequently [only one EST (AA259833) in dbEST was similarly spliced] and thus probably plays no physiological role. Further studies are planned to determine molecular basis for the dominant negative activity of Igf2sv. 6B72 cells were highly resistant to reovirus infection as determined by virus yield and cell survival at different times post-infection. Virus resistance was a genetically selected trait manifested by clonally pure cell populations, could be conferred by enforced IGF-II expression, and involved decreases in virus disassembly. Although decreased virus disassembly is sufficient to explain virus resistance, we do not exclude the possibility that other mechanisms may contribute to the resistance of 6B72 cells, since any genetic selection may generate clones with multiple, independent mutations. The fact that an early step in infection (uptake/disassembly) is defective in 6B72 cells makes it very difficult to test whether downstream steps might also be affected. While the original impetus of our studies was to understand the replication cycle of intracellular pathogens that cause acute and chronic infectious diseases, the finding of cell growth phenotypes associated with virus resistance is of some interest. It has been proposed that lytic viruses may used to treat certain malignancies [42-45]. However, based upon our observations, such therapy may carry a risk associated with selection of virus resistant cell clones with enhanced growth/survival potential. Additionally, chronic infections contribute to the development of a number of human cancers [46-49]. While the carcinogenic process is not well understood, cell proliferation associated with inflammation is also thought to contribute to tumour promotion [50,51]. The present study illustrates how carcinogenesis could also be influenced by selection for virus-resistant cells with mutations in genes affecting cell proliferation or survival. Conclusions This is the first reported use of gene entrapment to identify host genes affecting the susceptibility of cells to virus infection. These results indicate that IGF-II, and by inference other components of the IGF-II signaling pathway, can confer high levels of resistance to lytic reovirus infection. IGF-II expression specifically blocked virus disassembly. Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo. Therefore, these results illustrate a potential indirect mechanism of viral carcinogenesis by which cells selected to virus resistance may also have enhanced oncogenic potential. Methods Entrapment mutagenesis and selection of reovirus resistant clones To identify genes required for reovirus lytic infection, a gene trap retrovirus shuttle vector, U3NeoSV1, was used to generate mutagnized rat intestinal (RIE-1) cells [52]. RIE-1 cells were infected with the gene trap vector at a multiplicity of infection <0.1, and were selection in media containing G418 sulfate (0.7 mg/ml) (Clontech, Palo Alto, CA, USA)[52]. Twenty libraries of mutant RIE-1 cells, each consisting of 104 independent gene entrapment events, were generated and expanded until each mutant clone was represented by approximately 103 sibling cells. These cells were plated at low density and incubated in serum-free media for 3 days until they became quiescent, infected overnight with reovirus serotype 1 at a multiplicity of infection of 30 plaque forming units (pfu) per cell. The infected cells were detached with trypsin, DMEM medium containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT, USA) was added and the cells were allowed to reattach. After 4–6 hours the medium was replaced with serum-free medium and the cells were incubated for several days until only a few cells remained attached to the culture flask. Cells that survived the selection were allowed to form colonies that were expanded for further analysis. Reovirus stocks and infectivity assays Reovirus type 1 (Lang) and reovirus type 3 (Dearing) were previously described [53]. A stock of reovirus that was passaged twice in L cells was purified [14] and the purified virus band was fluorescein labelled as previously described [25]. For some experiments the top component, consisting of virus particles that are devoid of genome, was used to study the entry pathway [54]. Survival of parental L- and RIE-1 cells and RIE-1 and L-cells transfected with Igf2 constructs was determined in 96-well plates seeded at 5 × 104 per well. On the following day, serial dilutions of reovirus type 1 or type 3 were added in 100 μl of media and cells were incubated at 37°C and 5%CO2 for 1 hour. Cells were washed three times in PBS, and fresh media was added containing 0.1% anti-reovirus antibodies to inhibit secondary infection. Cells were incubated for 4 to 5 days, and surviving cells visualized with gentian violet. Studies were repeated a minimum of three times. To determine the titre of reovirus present in cells, cells were frozen and thawed three times and plaque assays were performed as previously described [14]. Titres of virus were repeated twice. Fluorescence microscopy For experiments involving fluorescein labeled reovirus, cells were grown on glass slides and fixed following appropriate times with 4% paraformaldehyde, dehydrated, and mounting in cytoseal acrylic resin (Stephens Scientific, Cornwall NJ, USA) to improve clarity and prevent bleaching. Fluorescence microscopy was performed using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY, USA), with a 40×/1.3 plan Neofluar objective and fluorescein filter set. Images were captured with a low-light, cooled CCD camera (Micromax, Photometries, Inc., Tucson, AZ, USA). Identification of genes disrupted by gene entrapment To identify the gene disrupted by the vector in clones surviving reovirus infection, the shuttle-vector property of U3NeoSV1 was utilized. Regions of genomic DNA adjacent to the U3NeoSV1 provirus were cloned by plasmid rescue, and sequenced [52]. Sequencing was done using an automated sequencer (ABI 3700 DNA Analyzer, Applied Biosystems, Foster City, CA, USA), and the results obtained were compared to databases available in the public domain (BLAST nr, est, and hgts). The probability of a match to sequences in the databases occurring by chance alone varies due to interspecies conservation and the length of the match. Matches to characterized genes were considered significant if the interspecies matches had a probability score p <10-5 and involved non-repetitive sequences. As indicated, virtually all of the genes identified had matches to murine or human gene sequences with p < 10-10 and rat with p < 10-20. Igf2 expression was assessed by northern blot hybridization Total RNA was isolated from cultured cells using Trizole reagent (Gibco BRL, Gaithersburg, MD, USA). 5 μg of RNA was separated on 1.2% agarose gel, and transferred to a nitrocellose membrane. Membranes were hybridized with random prime-labeled (Strategene, Cedar Creek, TX, USA) probes corresponding to a full length of Igf2 cDNA and either glyceraldehyde dehydrogenase (GAPDH) or β-actin cDNA. Igf2 cDNA isolation and expression Rat Igf2 cDNAs were obtained using reverse transcriptase PCR (RT-PCR). Total RNA was extracted from RIE and 6B72 cells using Trizole reagent (Life Technologies, Rockville, MD, USA). RT was performed on 1 μg of total RNA (PTC-100 programmable Thermal Controller, MJ Research. Inc, Watertown, MA, USA). A pair of primers was designed according to rat sequences: CTTCCAGGTACCAATGGGGATC (forward) and TTTGGTTCACTGATGGTTGCTG (reverse). A 500 bp DNA was amplified under following conditions: 95°C, 1 min; 40 cycles of 95°C 30 seconds, 60°C 30 seconds and 68°C 3 minutes; 68°C 10 min; 4°C. Immunoblotting analysis Cells were washed with PBS and lysed in SDS Lamelli buffer. Protein concentration was determined using the bicinchoninc acid protein assay (Sigma-Aldrich Corp., St. Louis, MO, USA). 20 μg of protein extract was loaded in each lane of a 10% SDS-PAGE and run at 100 V. Protein was transferred to a nitrocellulose membrane at 22 V overnight at 4°C. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membrane was then incubated with anti-mouse, CTCF (1:500, BD Transduction laboratories) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) in blocking buffer overnight at 4°C. Following 3 washes, the membranes were incubated with goat anti-mouse secondary antibody (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washings. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Transfection Cells were cultured to semi-confluence and plasmids expressing wild type and variant IGF-II transcripts were transfected into RIE-1 and 6B72 or L-cells using SuperFect Reagent (Qiagen, Inc. Valencia, CA, USA) according to the manufacturer's protocol. After 48 hours, transfected cells were passaged, 1:10, into medium containing hygromicin B (selective medium) at a concentration determined to kill 100% of non-transfected cells (150 mg/ml for RIE-1 and 6B72 cells, 650 mg/ml for L-cells). Cells were maintained in selective medium until clones appeared. Soft agar colony forming assay Dual layers of sea plaque agarose were made with the bottom layer consisting of a 50:50 mixture of 1.6% agarose solution 1:1 and 2X medium. The bottom layer was allowed to set for 4 hours, and then a 50:25:25 solution consisting of 2X medium, 1.6% stock agarose, and 1X medium containing cells, at a final concentration of 5000 cells/ml, was vortexed in a conical tube, and 2 ml was added to each well. Following 30 minutes at room temperature to allow the upper layer to set, plates were incubated at 37°C, 5% CO2 incubator for 7–10 days and checked for colony formation by microscopy. Analysis of viral protein expression in infected cells Cells were plated at 1.5 × 106 per well in 2 ml of medium in 6-well plates and allowed to sit over night. Cells were washed with phosphate-buffered saline (PBS), pH 7.4, and then infected with reovirus type 1 at the specified MOI. Virus was allowed to adsorb to cells for 1.5 hours at 4°C, washed twice with serum-free medium and incubated at 37°C and 5% CO2. At the indicated times, cells were scraped and lysed in Tris lysis buffer (10 mM Tris [pH 7.5], 2.5 mM MgCI2, 100 mM NaCI, 0.5% Triton x-100, 1 tablet Protease Inhibitor Cocktail Tablets [Roche Applied Science, Indianapolis, IN, USA] per 10 ml). After 30 min on ice, Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) were added to cell lysate samples (1:1). Protein samples were loaded in a 12% SDS-PAGE gel and run at 100 V. Protein was transferred to a nitrocellulose membrane at 100 V for 1 hour on ice. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membranes were then incubated with rabbit anti-reovirus type 1 (1:50) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) antibodies in blocking buffer overnight at 4°C. Following 3 washes in TBST, the membranes were incubated with goat anti-rabbit (for reovirus) or goat anti-mouse (β-actin) secondary antibodies (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washes. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Cell Proliferation Assay RIE-1, 6B72, or Igf2 transfected RIE-1 or 6B72 cells were seeded at 5 × 104 per well in 96-well plates, incubated at 37°C and 5% CO2. At 4, 6, 18, 48 hours post plating, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS], and an electron coupling reagent (phenazine methosulfate, [PMS]) were added at 20 μl per well (CellTiter 96 Aqueous Non-Radioactiver Cell Proliferation Assay, Promega, Madison, Wl). Plates were incubated for 2 hours, and then the absorbance was determined at 490 nm. Each set of conditions was repeated in triplicate. Authors' contributions JS, ELO, and CH conducted most of the laboratory work. KSW assisted in the analysis of fluorescein-labelled virus preparations. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones. HER and DHR provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript. Acknowledgements This work was supported by Public Health Service Grants (R01HG00684 to HER, RO1CA682383 to DHR), by a grant from the Kleberg Foundation, partially supported by Cancer Centre (Core) grant P30CA42014, and Avatar BioSci, Inc. Fluorescence microscopy was performed, in part, through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404 and HD15052. We would like to thank J. Hawiger, T. Hodge, and E. Eisenberg for review of the manuscript. B. Mooneyhan provided expert secretarial assistance. ==== Refs Arbour N Cote G Lachance C Tardieu M Cashman NR Talbot PJ Acute and persistent infection of human neural cell lines by human coronavirus OC43 J Virol 1999 73 3338 3350 10074188 Kennedy PG Clements GB Brown SM Differential susceptibility of human neural cell types in culture to infection with herpes simplex virus Brain 1983 106 101 119 6831193 Tozzi V Britton S Ehrnst A Lenkei R Strannegard O Persistent productive HIV infection in EBV-transformed B lymphocytes J Med Virol 1989 27 19 24 2537875 Montgomery LB Kao CY Verdin E Cahill C Maratos-Flier E Infection of a polarized epithelial cell line with wild-type reovirus leads to virus persistence and altered cellular function J Gen Virol 1991 72 2939 2946 1765767 Morgan DO Edman JC Standring DN Fried VA Smith MC Roth RA Rutter WJ Insulin-like growth factor II receptor as a multifunctional binding protein Nature 1987 329 301 307 2957598 10.1038/329301a0 Tong PY Tollefsen SE Kornfeld S The cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II J Biol Chem 1988 263 2585 2588 2963812 Bakay M Zhao P Chen J Hoffman EP A web-accessible complete transcriptome of normal human and DMD muscle Neuromuscul Disord 2002 12 S125 141 12206807 10.1016/S0960-8966(02)00093-7 Lobanenkov VV Nicolas RH Adler VV Paterson H Klenova EM Polotskaja AV Goodwin GH A novel sequence-specific DMA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene Oncogene 1990 5 1743 1753 2284094 Ahmed R Graham AF Persistent infections in L cells with temperature-sensitive mutants of reovirus J Virol 1977 23 250 262 886650 Ahmed R Canning WM Kauffman RS Sharpe AH Hallum JV Fields BN Role of the host cell in persistent viral infection: coevolution of L cells and reovoirus during persistent infection Cell 1981 25 325 332 7285112 10.1016/0092-8674(81)90050-7 Blay J Brown KD Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells J Cell Physiol 1985 124 107 112 3876338 Blay J Brown KD Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine Biochem J 1985 225 85 94 2983665 Organ EL Nalbantyan CD Nanney LB Woodward SC Sheng J Dubois RN Price J Sutcliffe M Coffey RJ Rubin DH Effects of transforming growth factor-alpha (TGF-alpha) in vitro and in vivo on reovirus replication DNA Cell Biol 2004 23 430 441 15294092 10.1089/1044549041474751 Rubin DH Kornstein MJ Anderson AO Reovirus serotype 1 intestinal infection: a novel replicative cycle with ileal disease J Virol 1985 53 391 398 2982027 Rubin DH Reovirus serotype 1 binds to the basolateral membrane of intestinal epithelial cells Microb Pathog 1987 3 215 219 3504220 10.1016/0882-4010(87)90098-2 von Melchner H Reddy S Ruley HE Isolation of cellular promoters by using a retrovirus promoter trap Proc Natl Acad Sci USA 1990 87 3733 3737 2339116 Hark AT Schoenherr CJ Katz DJ Ingram RS Levorse JM Tilghman SM CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus Nature 2000 405 486 489 10839547 10.1038/35013106 Bell AC Felsenfeld G Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene Nature 2000 405 482 485 10839546 10.1038/35013100 Dull TJ Gray A Hayflick JS Ullrich A Insulin-like growth factor II precursor gene organization in relation to insulin gene family Nature 1984 310 777 781 6382022 Buchanan CM Phillips AR Cooper GJ Preptin derived from proinsulin-like growth factor II (proIGF-ll) is secreted from pancreatic islet beta-cells and enhances insulin secretion Biochem J 2001 360 431 439 11716772 10.1042/0264-6021:3600431 Geuze HJ Slot JW Strous GJ Hasilik A von Figura K Possible pathways for lysosomal enzyme delivery J Cell Biol 1985 101 2253 2262 2933416 10.1083/jcb.101.6.2253 Johnson KF Kornfeld S A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function J Biol Chem 1992 267 17110 17115 1324923 Ludwig T Griffiths G Hoflack B Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells J Cell Biol 1991 115 1561 1572 1661730 10.1083/jcb.115.6.1561 De Ceuninck F Poiraudeau S Pagano M Tsagris L Blanchard O Willeput J Corvol M Inhibition of chondrocyte cathepsin B and L activities by insulin-like growth factor-ll (IGF-II) and its Ser29 variant in vitro: possible role of the mannose 6-phosphate/IGF-ll receptor Mol Cell Endocrinol 1995 113 205 213 8674828 10.1016/0303-7207(95)03632-H Georgi A Mottola-Hartshorn C Warner A Fields B Chen LB Detection of individual fluorescently labeled reovirions in living cells Proc Natl Acad Sci USA 1990 87 6579 6583 2118653 Nakagawa H Chadwick RB Peltomaki P Plass C Nakamura Y de La Chapelle A Loss of imprinting of the insulin-like growth factor II gene occurs by biallelic methylation in a core region of H19-associated CTCF-binding sites in colorectal cancer Proc Natl Acad Sci USA 2001 98 591 596 11120891 10.1073/pnas.011528698 Rainier S Johnson LA Dobry CJ Ping AJ Grundy PE Feinberg AP Relaxation of imprinted genes in human cancer Nature 1993 362 747 749 8385745 10.1038/362747a0 Wright JF Kurosky A Wasi S An endothelial cell-surface form of annexin II binds human cytomegalovirus Biochem Biophys Res Commun 1994 198 983 989 8117306 10.1006/bbrc.1994.1140 Zhao WQ Chen GH Chen H Pascale A Ravindranath L Quon MJ Alkon DL Secretion of Annexin II via activation of insulin receptor and insulin-like growth factor receptor J Biol Chem 2003 278 4205 4215 12431980 10.1074/jbc.M210545200 Zhu Z Gershon MD Ambron R Gabel C Gershon AA Infection of cells by varicella zoster virus: inhibition of viral entry by mannose 6-phosphate and heparin Proc Natl Acad Sci USA 1995 92 3546 3550 7724595 Brunetti CR Burke RL Kornfeld S Gregory W Masiarz FR Dingwell KS Johnson DC Herpes simplex virus glycoprotein D acquires mannose 6-phosphate residues and binds to mannose 6-phosphate receptors J Biol Chem 1994 269 17067 17074 8006011 Visser M Sijmons C Bras J Arceci RJ Godfried M Valentijn LJ Voute PA Baas F Allelotype of pediatric rhabdomyosarcoma Oncogene 1997 15 1309 1314 9315099 10.1038/sj.onc.1201302 Lynch CA Tycko B Bestor TH Walsh CP Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation Mol Cancer 2002 1 2 12234381 10.1186/1476-4598-1-2 Rainier S Dobry CJ Feinberg AP Loss of imprinting in hepatoblastoma Cancer Res 1995 55 1836 1838 7728748 Cui H Onyango P Brandenburg S Wu Y Hsieh CL Feinberg AP Loss of imprinting in colorectal cancer linked to hypomethylation of H19 and IGF2 Cancer Res 2002 62 6442 6446 12438232 McCann AH Miller N O'Meara A Pedersen I Keogh K Gorey T Dervan PA Biallelic expression of the IGF2 gene in human breast disease Hum Mol Genet 1996 5 1123 1127 8842730 10.1093/hmg/5.8.1123 De Souza AT Yamada T Mills JJ Jirtle RL Imprinted genes in liver carcinogenesis Faseb J 1997 11 60 67 9034167 Yun K Soejima H Merrie AE McCall JL Reeve AE Analysis of IGF2 gene imprinting in breast and colorectal cancer by allele specific- J Pathol 1999 187 518 522 10398115 10.1002/(SICI)1096-9896(199904)187:5<518::AID-PATH276>3.0.CO;2-3 Song BC Chung YH Kim JA Lee HC Yoon HK Sung KB Yang SH Yoo K Lee YS Suh DJ Association between insulin-like growth factor-2 and metastases after transcatheter arterial chemoembolization in patients with hepatocellular carcinoma: a prospective study Cancer 2001 91 2386 2393 11413529 10.1002/1097-0142(20010615)91:12<2386::AID-CNCR1272>3.3.CO;2-W Kawamoto K Onodera H Kondo S Kan S Ikeuchi D Maetani S Imamura M Expression of insulin-like growth factor-2 can predict the prognosis of human colorectal cancer patients: correlation with tumor progression, proliferative activity and survival Oncology 1998 55 242 248 9560057 10.1159/000011858 Duguay SJ Jin Y Stein J Duguay AN Gardner P Steiner DF Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis J Biol Chem 1998 273 18443 18451 9660813 10.1074/jbc.273.29.18443 Hirasawa K Nishikawa SG Norman KL Alain T Kossakowska A Lee PW Oncolytic reovirus against ovarian and colon cancer Cancer Res 2002 62 1696 1701 11912142 Norman KL Coffey MC Hirasawa K Demetrick DJ Nishikawa SG DiFrancesco LM Strong JE Lee PW Reovirus oncolysis of human breast cancer Hum Gene Ther 2002 13 641 652 11916487 10.1089/10430340252837233 Zwiebel JA Cancer gene and oncolytic virus therapy Semin Oncol 2001 28 336 343 11498828 10.1053/sonc.2001.26145 Norman KL Lee PW Reovirus as a novel oncolytic agent J Clin Invest 2000 105 1035 1038 10772645 Koike K Tsutsumi T Fujie H Shintani Y Kyoji M Molecular mechanism of viral hepatocarcinogenesis Oncology 2002 62 29 37 11868782 10.1159/000048273 Yee C Krishnan-Hewlett I Baker CC Schlegel R Howley PM Presence and expression of human papillomavirus sequences in human cervical carcinoma cell lines Am J Pathol 1985 119 361 366 2990217 Chang Y Cesarman E Pessin MS Lee F Culpepper J Knowles DM Moore PS Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma Science 1994 266 1865 1869 7997879 Hirshaut Y Cohen MH Stevens DA Epstein-Barr-virus antibodies in American and African Burkitt's lymphoma Lancet 1973 2 114 116 4124041 10.1016/S0140-6736(73)93061-4 Nomura A Stemmermann GN Chyou PH Kato I Perez-Perez Gl Blaser MJ Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii N Engl J Med 1991 325 1132 1136 1891021 McNamara D O'Morain C Helicobacter pylori and gastric cancer Ital J Gastroenterol Hepatol 1998 30 S294 298 10077758 Hicks GG Shi EG Li XM Li CH Pawlak M Ruley HE Functional genomics in mice by tagged sequence mutagenesis Nat Genet 1997 16 338 344 9241269 10.1038/ng0897-338 Fields BN Greene Ml Genetic and molecular mechanisms of viral pathogenesis: implications for prevention and treatment Nature 1982 300 19 23 6290894 Smith RE Zweerink HJ Joklik WK Polypeptide components of virions, top component and cores of reovirus type 3 Virology 1969 39 791 810 4311639 10.1016/0042-6822(69)90017-8

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==== Front BMC GenetBMC Genetics1471-2156BioMed Central London 1471-2156-5-251533314210.1186/1471-2156-5-25Research ArticleMolecular clock in neutral protein evolution Wilke Claus O [email protected] Keck Graduate Institute of Applied Life Sciences, 535 Watson Drive, Claremont, California 91711, USA2 Digital Life Laboratory, California Institute of Technology, Mail Code 136-93, Pasadena, California 91125, USA2004 27 8 2004 5 25 25 9 6 2004 27 8 2004 Copyright © 2004 Wilke; licensee BioMed Central Ltd.2004Wilke; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A frequent observation in molecular evolution is that amino-acid substitution rates show an index of dispersion (that is, ratio of variance to mean) substantially larger than one. This observation has been termed the overdispersed molecular clock. On the basis of in silico protein-evolution experiments, Bastolla and coworkers recently proposed an explanation for this observation: Proteins drift in neutral space, and can temporarily get trapped in regions of substantially reduced neutrality. In these regions, substitution rates are suppressed, which results in an overall substitution process that is not Poissonian. However, the simulation method of Bastolla et al. is representative only for cases in which the product of mutation rate μ and population size Ne is small. How the substitution process behaves when μNe is large is not known. Results Here, I study the behavior of the molecular clock in in silico protein evolution as a function of mutation rate and population size. I find that the index of dispersion decays with increasing μNe, and approaches 1 for large μNe . This observation can be explained with the selective pressure for mutational robustness, which is effective when μNe is large. This pressure keeps the population out of low-neutrality traps, and thus steadies the ticking of the molecular clock. Conclusions The molecular clock in neutral protein evolution can fall into two distinct regimes, a strongly overdispersed one for small μNe, and a mostly Poissonian one for large μNe. The former is relevant for the majority of organisms in the plant and animal kingdom, and the latter may be relevant for RNA viruses. ==== Body Background Kimura has argued that the majority of nucleotide substitutions that accumulate in genes over time are selectively neutral, and go to fixation purely by chance [1]. One major prediction of Kimura's neutral theory is that the substitution process should be a Poisson process, with the mean number of substitutions per unit time equal to the variance. In contrast to this theory, empirical studies often find the variance to be significantly larger than the mean [2-8]. This observation has been termed the "overdispersed molecular clock". (For an excellent review of both empirical evidence and mathematical theories, see Ref. [9].) It is possible to reconcile Kimura's theory with the overdispersed molecular clock via Takahata's fluctuating neutral space model [10-12]. If the neutral mutation rate fluctuates slowly, then the substitution process ceases to be Poissonian, and becomes indeed overdispersed. However, the problem with the fluctuating neutral space model is that it does not offer any argument for why the neutral mutation rate should fluctuate, and thus ultimately fails to explain the observed substitution patterns. An explanation for fluctuations in neutral mutation rate was recently proposed by Bastolla et al. [13-16]. Different proteins with identical structure naturally vary in their neutrality, that is, in the fraction of single-point mutants that are viable. Therefore, as a gene slowly drifts through sequence space, the neutral mutation rate will fluctuate in correlation to the changing neutrality, and this fluctuation alone could be sufficient to explain the overdispersed molecular clock. Bastolla et al. studied the substitution process in a variety of models of neutral protein evolution in silico, and found significant overdispersion in all cases they considered. However, the simulations that Bastolla et al. carried out were limited to cases in which the product of mutation rate μ and population size Ne is small (because Bastolla et al. used only a single sequence as the representative of the whole population, an approach that is justified for μNe ≲ 1). Since population size and mutation rate can be substantial in some species (most notably in RNA viruses), it is justified to ask how general this result is for arbitrary values of μNe. It is known that large populations and populations evolving under high mutation pressure experience a strong selective pressure to avoid regions of low neutrality, an effect that has been termed "evolution of mutational robustness" [17-20]. In equilibrium, such populations settle in areas of sequence space that have above-average neutrality. As a result, regions of low neutrality are not represented in the population, and the distribution of neutralities in the population is much narrower than the total distribution of neutralities in sequence space. Therefore, we should expect that the neutral mutation rate does not fluctuate strongly under these conditions, and that the molecular clock will not be significantly overdispersed. For the present paper, I have studied the behavior of the substitution process under neutral protein evolution as a function of mutation rate μ and population size Ne. I have found that the accumulation of non-synonymous mutations is substantially overdispersed for small μNe, in agreement with the results of Bastolla et al., but approaches a Poisson process when μNe ≫ 1. The accumulation of synonymous substitutions is always Poissonian, regardless of the value of μNe. Results I carried out simulations with DNA sequences of length L = 75. I determined the fitness of a DNA sequence by translating it into the corresponding amino-acid sequence, and determining its native fold within the framework of a lattice-protein model. (A sequence would have fitness 1 if it folded into a pre-determined target structure, and fitness 0 otherwise.) I used a simple model of maximally compact proteins on a 5 × 5 lattice. This protein-folding model is much simpler than the ones used by Bastolla et al. [13,14], but has been shown to produce realistic distributions of folding free energies and neutralities [21-23]. The advantage of the simpler model is that entire populations of evolving sequences can be simulated, instead of just individual sequences. First, I have found that my model produces overdispersion (that is, an index of dispersion R substantially above 1) for non-synonymous substitutions, but not for synonymous substitutions. The finding for synonymous mutations is not surprising, because changes in the protein's neutrality do not affect the probability with which a synonymous mutation is neutral (which is always one). Neutral evolution could produce overdispersion in the synonymous substitutions only if the number of synonymous sites in the sequence were undergoing significant fluctuations. While these fluctuations do occur, they are apparently not large enough to affect the index of dispersion. Second, I have found that for non-synonymous substitutions, R decays quickly with increasing population size Ne at fixed μ (Fig. 1). Since one reason for a decaying index of dispersion could be a reduced number of accumulated mutations, I have studied how the mean number of accumulated mutations behaves as a function of population size. Instead of staying constant or decreasing, the mean increases with increasing Ne, while the variance decreases (Fig. 2). This result shows that the reduction in R is not caused by a mere reduction in the accumulated mutations, and that the substitution process does indeed shift from overdispersed to Poissonian as the population size increases. Figure 1 Index of dispersion as a function of population size Ne for synonymous and non-synonymous substitutions (τ = 1000, μ = 0.075). Figure 2 Mean, and variance, of lineage-adjusted number of non-synonymous substitutions as a function of population size Ne (τ = 1000, μ = 0.075). Quantities were calculated from all 500 replicates at each population size. For non-synonymous substitutions, R decays with Ne because of evolution of mutational robustness. However, mutational robustness is caused by large μNe, rather than large Ne alone, and the parameter region in which mutational robustness becomes relevant is μNe ≳ 10 [17]. Therefore, it is more instructive to plot R as a function of μNe. The only problem with a naive plot of that sort is that R increases as a function of μτ, where τ is the length of the time window during which mutations accumulate [9]. Thus, in Fig. 3, I show R for constant μτ as a function of μNe. Note that in this figure, instead of the sequence-wide mutation rate μ, I use the non-synonymous mutation rate μn = 0.76 μ, which is corrected for the fact that only approximately 76% of mutations hit non-synonymous sites. (76% is the expected fraction of non-synonymous sites in a random DNA sequence.) Figure 3 shows that the transition from an overdispersed to a Poissonian substitution process occurs for μNe between approximately 10 and 100, in agreement with Ref. [17], and that the transition region seems to be largely independent of the value of μτ. Figure 3 Index of dispersion for non-synonymous mutations as a function of the product of non-synonymous mutation rate μn (= 0.76 μ) and population size Ne. Figure 3 also shows that R increases with μτ. This dependency becomes clearer in Fig. 4, where I display R as a function of μτ for fixed μNe. The figure shows substantial increase in R with increasing μτ for small to moderate μNe. However, even for μNe well above 50, there is still a slight increase in R with μτ. Therefore, my results do not settle the question of whether the substitution process becomes truly Poissonian for sufficiently large μNe, or whether it just approaches a Poisson process but always remains slightly overdispersed. To settle this question, one would have to carry out simulations with much larger τ and Ne. Unfortunately, the protein folding model I use is still too computationally intensive to permit such simulations with current computational resources. Figure 4 Index of dispersion for non-synonymous mutations as a function of the product of non-synonymous mutation rate μn (= 0.76 μ) and divergence time τ. Discussion My results show that the size of the product μNe has a substantial effect on the index of dispersion under neutral evolution. The substitution process is strongly overdispersed for small μNe, but approaches a Poisson process as μNe grows large. Therefore, the next question is which of the two regimes has more biological relevance. As discussed by Cutler [9], the biggest problem in explaining the overdispersed molecular clock is not to come up with mechanisms that produce overdispersion, but to find a general mechanism that does not depend on special conditions or finely-tuned parameters. To assess the likelihood that fluctuations in neutrality contribute to the overdispersed molecular clock, we have to know the mutation rate and population size for the species of interest. It is notoriously difficult to obtain accurate data for these parameters, and only a few species have been studied in depth. One of the best data sets available is probably the one for Drosophila. Keightley and Eyre-Walker estimated the per-nucleotide substitution rate in Drosophila to be u = 2.2 × 10-9 [24]. If we assume that the average gene in Drosophila is 1770 bp long [24], and that 76% of the nucleotides are non-synonymous (this number stems from averaging the number of non-synonymous sites over all codons with equal weight), then the average number of non-synonymous sites per gene is 1345 bp. Thus, the average rate of non-synonymous mutations per gene is μn = 3.0 × 10-6. With an effective population size of approximately 3 × 105 [25], we get a product of population size and per-gene-non-synonymous mutation rate of approximately 1. Since selection for mutational robustness starts to take effect when this product is substantially larger than 1, Drosophila lies well within the parameter region in which we expect overdispersion to be caused by neutral evolution. For other higher organisms, in particular mammals, which tend to have comparatively small population sizes, we can expect that the product μnNe falls into the same parameter region. On the other hand, for microorganisms, which can have very large population sizes, mutational robustness may play a role in their evolution. In particular, RNA viruses have genomic mutation rates on the order of one [26,27] and their genomes consist typically of only a handful of genes. Because RNA viruses undergo severe bottlenecks on a regular basis, their effective population size Ne is much smaller than the number of virus particles in infected individuals (which can exceed 1012), and is more closely related to the number of infected individuals. For HIV-1, Ne has been estimated to be approximately 102 for subtype A, and 105 for subtype B [28]. The preceeding paragraph shows that neutral evolution of proteins is probably one source of overdispersed non-synonymous substitutions in Drosophila and other organisms. However, overdispersion has been observed in synonymous substitutions as well. For example, Zeng et al. [29] found an index of dispersion R significantly above one for synonymous, but not for non-synonymous substitutions in Drosophila. For mammals, some studies found R significantly above one for both synonymous and non-synonymous substitutions [8], while others found only the non-synonymous substitution process to be overdispersed [30]. Therefore, it is likely that other processes than neutral protein evolution also contribute to overdispersion. Such processes can be selection for optimal codon usage in the case of synonymous mutations, and positive selection on the amino acid level in the case of non-synonymous mutations. I have demonstrated that large μNe results in a substitution process with little overdispersion. However, I have not yet given an explanation for how overdispersion is reduced in populations with large μNe. There are two elements: First, selection for mutational robustness reduces the fraction of sequences with low neutrality, and increases the fraction of sequences with high neutrality, thus making the population more homogeneous and reducing the overall range of neutralities [17-20]. Second, a sequence with low neutrality will experience a real selective disadvantage in comparison to a sequence with high neutrality for large μNe, and will therefore have a reduced probability to end up on the line of descent. While this selective disadvantage is often small, it can nevertheless determine the evolutionary fate of a sequence in a large population. The larger the population, the more sensitive it becomes to small fitness differences, so that in a very large population a sequence with only a moderate reduction in neutrality will have a small probability to end up on the line of descent. (The fact that the mean substitution rate increases with the population size, as seen in Fig. 2, is also consistent with this reasoning. The larger the population size, the more high-neutrality sequences end up on the line of descent, which is reflected in the increase in the mean substitution rate.) Throughout this paper, I have considered only neutral or lethal mutations. It is a reasonable question to ask if and how deleterious mutations would change my results. The answer is that they probably have only a minor impact, and the less so the larger Ne, unless they are very slightly deleterious. In order to affect the molecular clock, the deleterious mutations must end up on the line of descent, that is, they must go to fixation. The probability of fixation pfix of deleterious mutants drops exponentially with the population size, pfix = [1 - exp(2s)]/ [1 - exp(2sNe)], where s is the selective disadvantage of the deleterious mutation [31]. Therefore, for reasonable population sizes, only very slightly deleterious mutations can go to fixation and thus affect the molecular clock. This reasoning is independent of the size of μNe, as long as Ne is large in comparison to s. Conclusions The present study supports the following conclusions: • Neutral drift of proteins can lead to an overdispersed substitution process for non-synonymous mutations, but not for synonymous mutations. • The amount of overdispersion in the non-synonymous substitution process depends strongly on the product of mutation rate and population size. As this product increases, the substitution process becomes more and more Poissonian. The transition region starts at μNe ≈ 10, and extends to values well above 100. • It is not clear whether there are any species that have a sufficiently large population size and mutation rate to prevent overdispersion through neutral drift. In Drosophila, the product of mutation rate and population size is close to one, which is well below the parameter region in which the substitution process turns Poissonian. Methods Lattice protein model I implemented a version of the 5 × 5 lattice protein model put forward by Goldstein and coworkers [21-23,32]. In this model, proteins are sequences of n = 25 residues that fold into a maximally compact structure on a two-dimensional grid of 5 × 5 lattice points. There are 1081 distinct possible conformations in this model, and the partition function can be evaluated exactly by summing over the contact energies of all distinct conformations. The contact energy of a conformation i is where is the contact energy between amino acids at location j and at location k in the sequence, and is 1 if the two amino acids are in contact in conformation k, and 0 otherwise. The partition function is where the sum runs over all 1081 conformations. A sequence folds into conformation f if the contact energy for that conformation is lower than the contact energies of all other formations, Ef <Ei for all i ≠ f, and if the free energy of folding, which is defined as is smaller than some cutoff ΔGcut. Throughout this study, I used kT = 0.6 and ΔGcut = 0. The contact energies where taken from Table VI in Ref. [33]. Sequence evolution I simulated the evolution of populations of DNA sequences in discrete, non-overlapping generations. Population size is denoted by Ne. The fitness of a sequence was 1 if the DNA sequence translated into a peptide sequence that could fold into a chosen target structure, and had a free energy of folding smaller than Gcut. Otherwise, the fitness of the sequence was 0. All sequences had length L = 75. In each successive generation, sequences with fitness 1 were randomly chosen to reproduce, until the new generation had Ne members. At reproduction, the sequences were mutated, with an average of μ base pair substitutions per sequence. I let each population evolve for several thousand generations, and kept track of the full genealogic information of all sequences in the population. In order to measure the molecular clock of fixed mutations only, I studied the pattern of base substitutions in a window of τ generations along the line of descent backwards in time, starting from the most recent common ancestor of the final population. I varied the parameters Ne (10, 33, 100, 330, 1000, 3300), μ (0.0075, 0.075, 0.75), and τ (500, 1000). For each set of parameters, I carried out 500 replicates (each with a different, randomly chosen target structure), to obtain a distribution for the number of synonymous and non-synonymous substitutions Sd and Nd. Since there was some variation in the number of synonymous and non-synonymous sites across different target structures (on the order of approximately ± 5% variation from the mean), I then applied a correction factor to Sd and Nd to bring them into comparable units: I calculated the corrected number of synonymous substitutions as Here, S is the mean number of synonymous sites for the given replicate, and (S) is the average of S over all 500 replicates. Likewise, I calculated (Indices of dispersion calculated without this correction factor are slightly larger than the ones reported here, because the variation in S and N creates additional variance in Sd and Nd). Similar correction factors have been used in sequence analysis [7], and are generally referred to as lineage adjustments. They control for differences among lineages that are primarily related to the expected number of substitutions in a lineage, and thus should not enter the index of dispersion. To obtain an estimate for mean and standard error of the index of dispersion, I subdivided the 500 results into 10 blocks of 50 each, and calculated mean and variance of the number of substitutions for each block. The ratio of variance to mean for a given set of substitutions (synonymous or non-synonymous) in a block is the index of dispersion for this data set. I then calculated mean and standard error for the index of dispersion from the individual results of the 10 blocks. The total CPU time needed to carry out all simulations was several months on a small cluster of Pentium II 500 MHz machines. Calculation of synonymous and non-synonymous substitutions and sites I calculated the number of synonymous and non-synonymous sites S and N and the number of synonymous and non-synonymous substitutions Sd and Nd according to the method proposed by Nei and Gojobori [34]. In short, under this method the number of synonymous sites si of a codon i is the fraction of possible substitutions to that codon that leave the residue unchanged. The number of non-synonymous sites ni for the same codon is ni = 3 - si. For the complete sequence, S and N are calculated as and where i runs over all codons in the sequence. The number of synonymous or non-synonymous substitutions sd,i or nd,i between two codons is the average number of such substitutions, where the average is taken over all paths that lead from one codon to the other. The total number of synonymous or non-synonymous substitutions between two sequences is the sum over all individual constributions, and (again, i runs over all codons in the sequence). To calculate the number of synonymous or non-synonymous substitutions along the line of descent, I simply summed up all synonymous or non-synonymous substitutions that occurred from generation to generation. Because the full evolutionary history was known, a correction for multiple mutations such as the Jukes-Cantor correction [35] was not necessary. I also averaged the number of synonymous and non-synonymous sites over all sequences along the line of descent, to get the mean number of synonymous and non-synonymous sites for the given evolutionary trajectory. Authors' contributions COW carried out all aspects of this study. Acknowledgements This work was supported in part by the NSF under Contract No. DEB-9981397. I thank Ugo Bastolla for helpful comments on an earlier version of this paper. ==== Refs Kimura M The Neutral Theory of Molecular Evolution 1983 Cambridge: Cambridge University Press Ohta T Kimura M On the constancy of the evolutionary rate of cistrons J Mol Evol 1971 1 18 25 4377445 Langley CH Fitch WM An estimation of the constancy of the rate of molecular evolution J Mol Evol 1974 3 161 177 4368400 Gillespie JH The molecular clock may be an episodic clock Proc Natl Acad Sci USA 1984 81 8009 8013 6595674 Gillespie JH Natural selection and the molecular clock Mol Biol Evol 1986 3 138 155 2832690 Gillespie JH Variability of evolutionary rates of DNA Genetics 1986 113 1077 1091 3744027 Gillespie JH Lineage effects and the index of dispersion of molecular evolution Mol Biol Evol 1989 6 636 647 2488476 Ohta T Synonymous and non-synonymous substitutions in mammalian genes and the nearly neutral theory J Mol Evol 1995 40 56 63 7714912 Cutler DJ Understanding the overdispersed molecular clock Genetics 2000 154 1403 1417 10757779 Takahata N On the overdispersed molecular clock Genetics 1987 116 169 179 3596230 Takahata N Statistical models of the overdispersed molecular clock Theor Popul Biol 1991 39 329 344 1896948 Cutler DJ The index of dispersion of molecular evolution: slow fluctuations Theor Popul Biol 2000 57 177 186 10792981 10.1006/tpbi.1999.1445 Bastolla U Roman HE Vendruscolo M Neutral evolution of model proteins: Diffusion in sequence space and overdispersion J Theor Biol 1999 200 49 64 10479539 10.1006/jtbi.1999.0975 Bastolla U Porto M Roman HE Vendruscolo M Lack of self-averaging in neutral evolution of proteins Phys Rev Lett 2002 89 20801 10.1103/PhysRevLett.89.208101 Bastolla U Porto M Roman HE Vendruscolo M Connectivity of neutral networks, overdispersion, and structural conservation in protein evolution J Mol Evol 2003 56 243 254 12612828 10.1007/s00239-002-2350-0 Bastolla U Porto M Roman HE Vendruscolo M Statistical properties of neutral evolution J Mol Evol 2003 57 S103 S119 15008407 10.1007/s00239-003-0013-4 van Nimwegen E Crutchfield JP Huynen M Neutral evolution of mutational robustness Proc Natl Acad Sci USA 1999 96 9716 9720 10449760 10.1073/pnas.96.17.9716 Bornberg-Bauer E Chan HS Modeling evolutionary landscapes: Mutational stability, topology, and superfunnels in sequence space Proc Natl Acad Sci USA 1999 96 10689 10694 10485887 10.1073/pnas.96.19.10689 Wilke CO Adaptive evolution on neutral networks Bull Math Biol 2001 63 715 730 11497165 10.1006/bulm.2001.0244 Wilke CO Adami C Evolution of mutational robustness Mutat Res 2003 522 3 11 12517406 10.1016/S0027-5107(02)00307-X Taverna DM Goldstein RA The distribution of structures in evolving protein populations Biopolymers 2000 53 1 8 10644946 10.1002/(SICI)1097-0282(200001)53:1<1::AID-BIP1>3.3.CO;2-O Taverna DM Goldstein RA Why are proteins so robust to site mutations? J Mol Biol 2002 315 479 484 11786027 10.1006/jmbi.2001.5226 Taverna DM Goldstein RA Why are proteins marginally stable? Proteins 2002 46 105 109 11746707 10.1002/prot.10016 Keightley PD Eyre-Walker A Deleterious mutations and the evolution of sex Science 2000 290 331 333 11030650 10.1126/science.290.5490.331 Li YJ Satta Y Takahata N Paleo-demography of the Drosophila melanogaster subgroup: application of the maximum likelihood method Genes Genet Syst 1999 74 117 127 10650839 10.1266/ggs.74.117 Drake JW Rates of spontaneous mutation among RNA viruses Proc Natl Acad Sci USA 1993 90 4171 4175 8387212 Drake JW Holland JJ Mutation rates among RNA viruses Proc Natl Acad Sci USA 1999 96 13910 13913 10570172 10.1073/pnas.96.24.13910 Grasslya NC Harveya PH Holmes EC Population dynamics of HIV-1 inferred from gene sequences Genetics 1999 151 427 438 9927440 Zeng LW Comeron JM Chen B Kreitman M The molecular clock revisited: the rate of synonymous vs. replacement change in Drosophila Genetica 1998 102/103 369 382 9720289 10.1023/A:1017035109224 Smith NG Eyre-Walker A Partitioning the variation in mammalian substitution rates Mol Biol Evol 2003 20 10 17 12519900 Kimura M On the probability of fixation of mutant genes in a population Genetics 1962 47 713 719 14456043 Buchler NEG Goldstein RA Effect of alphabet size and foldability requirements on protein structure designability Proteins 1999 34 113 124 10336377 10.1002/(SICI)1097-0134(19990101)34:1<113::AID-PROT9>3.3.CO;2-A Miyazawa S Jernigan RL Estimation of effective inter-residue contact energies from protein crystal structures: quasichemical approximation Macromolecules 1985 18 534 552 Nei M Gojobori T Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions Mol Biol Evol 1986 3 418 426 3444411 Jukes TH Cantor CR Munro HN Evolution of protein molecules In Mammalian protein metabolism III 1969 New York: Academic Press 21 132

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==== Front BMC Mol BiolBMC Molecular Biology1471-2199BioMed Central 1471-2199-5-161535021110.1186/1471-2199-5-16Research ArticleCoactivators p300 and PCAF physically and functionally interact with the foamy viral trans-activator Bannert Helmut [email protected] Walter [email protected] Vasily V [email protected] Yoshihiro [email protected]ügel Rolf M [email protected] Department of Retroviral Gene Expression, German Cancer Research Center, Applied Tumor Virology, Heidelberg, Germany2 Abteilung Virologie, Hygiene-Institut, Universität Heidelberg, 69120 Heidelberg, Germany3 André Lwoff Institut, CNRS UR079, 7 Rue Guy Moquet, Villejuif 94801, France4 Dana-Farber Cancer Institute, 44 Binney Street, Harvard Medical School, Boston, MA 02115, USA2004 6 9 2004 5 16 16 24 5 2004 6 9 2004 Copyright © 2004 Bannert et al; licensee BioMed Central Ltd.2004Bannert et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Foamy virus Bel1/Tas trans-activators act as key regulators of gene expression and directly bind to Bel1 response elements (BRE) in both the internal and the 5'LTR promoters leading to strong transcriptional trans-activation. Cellular coactivators interacting with Bel1/Tas are unknown to date. Results Transient expression assays, co-immunoprecipitation experiments, pull-down assays, and Western blot analysis were used to demonstrate that the coactivator p300 and histone acetyltransferase PCAF specifically interact with the retroviral trans-activator Bel1/Tas in vivo. Here we show that the Bel1/Tas-mediated trans-activation was enhanced by the coactivator p300, histone acetyltransferases PCAF and SRC-1 based on the crucial internal promoter BRE. The Bel1/Tas-interacting region was mapped to the C/H1 domain of p300 by co-immunoprecipitation and pull-down assays. In contrast, coactivator SRC-1 previously reported to bind to the C-terminal domain of p300 did not directly interact with the Bel1 protein but nevertheless enhanced Bel1/Tas-mediated trans-activation. Cotransfection of Bel1/Tas and p300C with an expression plasmid containing the C/H1domain partially inhibited the p300C-driven trans-activation. Conclusions Our data identify p300 and PCAF as functional partner molecules that directly interact with Bel1/Tas. Since the acetylation activities of the three coactivators reside in or bind to the C-terminal regions of p300, a C/H1 expression plasmid was used as inhibitor. This is the first report of a C/H1 domain-interacting retroviral trans-activator capable of partially blocking the strong Bel1/Tas-mediated activation of the C-terminal region of coactivator p300. The potential mechanisms and functional roles of the three histone and factor acetyltransferases p300, PCAF, and SRC-1 in Bel1/Tas-mediated trans-activation are discussed. ==== Body Background In the sequential model of transcriptional regulation including viral trans-activation, coactivators CBP/p300 require concerted action of multiple protein factors provided the nucleosomal structures allow access to the DNA template [1-3]. The factors that interact with coactivators encompass sequence-specific DNA binding activators, non-DNA binding coactivators, and essential components of the basal transcriptional machinery. Two large, closely related human proteins, p300 and CBP, were identified and shown to function as versatile signal integrators of many transcription factors to facilitate transcriptional activation or repression, and, in addition, as connectors of multiple transduction pathways. Both proteins contain several conserved domains that include three Cys/His-rich (C/H1, C/H2, and C/H3) domains, the histone acetyltransferase (HAT), KIX, and Gln-rich (Q) domains among others (Fig. 1) [2,4,5]. It is mainly due to these domains that a plethora of transcriptional activators interact with p300/CBP. Thus, p300/CBP coactivators act as a physical and functional scaffold or bridge between various cellular or viral trans-activators and the basal transcriptional machinery. Both proteins function by mediating positive or negative cross talk between different signaling pathways and participate in fundamental cellular processes that include embryonic development, cell growth, differentiation, and apoptosis. In addition, they can act as tumor suppressors and, last but not least, directly interact with diverse viral trans-activators to facilitate virus replication or viral activator-mediated transformation [6,7]. Figure 1 Coactivator p300 enhances Bel1/Tas-mediated trans-activation. Simplified schematic drawing of p300 domains and structure of deletion mutants (not drawn to scale). Characterized domains of p300 [2, 5] relevant for this report are highlighted by differently marked boxes: the FLAG epitope upstream of the N-terminus of p300 is marked by shaded boxes in constructs no. 1 through 8, the C/H1 and KIX domains by black and striped boxes, respectively. Numbers above the rectangles indicate the number of amino acids of p300 and its derivatives. The intrinsic HAT domain of p300 is depicted between the C/H2 and C/H3 domains; additional HATs PCAF and SRC-1 reported to associate with the distinct p300 regions [8, 24, 25] are indicated as rectangles above p300. The domain marked "Q" is the glutamine-rich domain of p300. Construct no. 5 has a deletion between the C/H1 and KIX domain (broken line). Constructs no. 9 and 10 contain the GST-C/H1 and GST-ΔC/H1-KIX fusion proteins; partial shadings mark GST regions. Several models have been proposed to explain transcriptional activation. Coordinated recruitment of coactivators by diverse transcriptional activators to specific promoter target sites has been shown by a collective effort of many groups [8]. According to the models, different coactivators either modify chromatin structure by altering the nucleosomal DNA thereby affecting its accessibility to DNA-binding proteins or, alternatively, form complexes with HAT activities that by acetylation of specific Lysines in histone N-terminal tails weaken interactions between DNA and the histone octamer. Moreover, the HAT activities of some coactivators acetylate non-histone substrates such as viral and cellular trans-activators, for instance p53 [9]. Diverse viral trans-activator proteins were found to interact with distinct domains of p300/CBP and PCAF. Prominent among them are the early adenoviral E1A antigen [10,11], Epstein-Barr virus protein EBNA-2 [12], and human T-cell leukemia virus oncoprotein Tax [13,14]. Several other modifications including methylation, phosphorylation, and ubiquitination lead to either diminished or increased DNA binding of the activators that, in turn, will result in either a repression or activation of gene expression. In addition, both coactivators were reported to interact with additional HAT enzymes, namely PCAF and SRC-1 [15,16]. The apparently nonpathogenic primate foamy viruses (PFV) show a wide host range and tissue tropism and have been developed into vectors that efficiently transduce SCID-repopulating cells [17]. The PFV Bel1/Tas protein has been characterized as a transcriptional trans-activator of the acidic class and is known to directly interact with its responsive elements (BRE) [18,19]. Bel1/Tas is a nuclear protein and acts as the key regulator absolutely required for virus replication. The minimal Bel1-specific DNA target site is 27 base-pairs long and located within the internal promoter (IP.BRE) upstream of the second cap site that is part and parcel of the second PFV transcription unit [20]. Additional Bel1/Tas DNA target sites in the LTR region of the PFV DNA genome were not analyzed in this study. The acidic trans-activation domain (TAD) was mapped to the C-terminus of Bel1 with little if any protein homology to other FV Bel1/Tas proteins from different species. Previously, we identified the nuclear factor 1 (NF1) as a repressor of Bel1/Tas-mediated trans-activation [21]. This repression was due to the fact that the specific family members NF1-C and -X interacted with parts of the IP.BRE and its flanking sequences. Since the NF1-mediated repression of the promoter of mouse mammary tumor virus was abrogated by distinct coactivators [22], we investigated which of the known coactivators and HAT proteins were capable of interacting with the PFV Bel1/Tas activator in the context of the IP.BRE promoter that is absolutely required for virus replication [20]. Here we report that the Bel1/Tas DNA binding protein functionally interacted with p300 and with the well-known HAT factor PCAF. In addition, SRC-1 enhanced Bel1/Tas trans-activation. This is the first time that these cellular coactivators have been shown to interact with the Bel/Tas1 trans-activator protein. Furthermore, Bel1/Tas binding to the C/H1 domain of p300 and coactivator-driven trans-activation seem to follow a unique pathway. Results Coactivator p300 enhances Bel1/Tas-mediated activation To examine whether p300 (Fig. 1, first line) enhanced the ability of Bel1 to trans-activate the Bel1 internal promoter (IP.BRE), transient reporter gene assays were performed. The IP.BRE that extends from -1 to -192 of the second cap site of the PFV genome was cloned into the pGL3-pro-luc reporter plasmid [21]. The results of the luc assays showed that full-length p300FL enhanced Bel1/Tas-mediated activation in an apparently nonlinear fashion (Fig. 2, upper left panel). To monitor the expression level of the p300 protein, Western blot analysis was carried out in parallel with increasing concentrations of the coactivator at fixed concentrations of the pbel1s expression plasmid that carries the retroviral trans-activator under the control of the CMV-IE promoter. The results of immunoblotting shown in the lower left panel of Figure 2 revealed that Bel1/Tas was expressed at similar levels, as expected, and p300FL expression levels proportional to the input. In parallel experiments, truncated p300 forms, p300N and p300M, were also assayed and yielded moderate levels of enhancement of Bel1/Tas-mediated activation lower than those of p300FL (Fig. 3). Unexpectedly, the most extensive level of enhancement of Bel1/Tas-induced activation was reached with the C-terminal region p300C (Fig. 2, upper right hand panel). Again, Western blot analysis of p300C showed that expression levels of p300C protein increased proportionately to the transfected plasmid DNA while Bel1/Tas expression levels were unchanged (Fig. 2, lower right panel). The precise boundaries of the three p300 versions used are shown in Figure 1. The p300 bands marked by arrows are likely due to modified p300 proteins that are known to be modified by phosphorylation, acetylation and sumoylation (2, 5, 26). We next sought to determine whether the enhancing effect of p300 on Bel1/Tas-mediated activation was due to a physical interaction with the Bel1/Tas protein. Figure 2 Coactivator p300 enhances Bel1/Tas-mediated activation. Transient expression assays were performed with pGL3-luc plasmids containing the internal PFV BRE promoter (-1 to -192) after transfection of the pCMV-bel1s expression plasmid alone or separate cotransfection with p300FL and p300C expression plasmids [4]. Normalized luc activities are shown as fold activation in upper panels (for details, see Methods). Expression levels of Bel1/Tas and p300 proteins after cotransfection of 293T cells with increasing p300FL and p300C DNA concentrations and pbel1s DNA of 1.0 μg. Aliquots of the cellular lysates used for luc assays shown in upper panel were in parallel subjected to immunoblot analysis with monoclonal antibody against the FLAG epitope of the p300FL protein (lower left panel), and against the p300C protein (lower right hand panel). Polyclonal antibody against Bel1 was used for Bel1/Tas expression (bottom lanes in lower panels). Figure 3 Reporter gene assays of shortened p300 expression plasmids. Transient expression assays were done as described under Fig. 2 except for that cotransfections were carried out with p300N and p300M plasmids; for boundaries of p300, see Fig. 1. Bel1/Tas interacts with p300 in vivo To examine whether the coactivator p300 interacts with the retroviral activator Bel1/Tas, binding of p300FL to the Bel1/Tas protein was analyzed by immune precipitation. 293T cells were cotransfected with the full-length p300FL and pbel1s expression plasmids and metabolically labeled with [35S]-Methionine and -Cysteine. Cellular lysates were precleared and subjected to immunoprecipitation with an antibody directed against the Bel1/Tas protein except for that in lane 1 (Fig. 4). After separation by SDS-PAGE and exposure, the resulting autoradiogram showed that the Bel1-specific antibody had effectively precipitated the p300FL-Bel1/Tas protein complex at both p300FL DNA concentrations of 10 μg (Fig. 4, lane 3) and 20 μg (lane 6). In the control where pbel1s was omitted the immune precipitation did not reveal any band comparable in size to p300 (lane 4). In the immunoprecipitation shown in lane 1 instead of an antibody against Bel1/Tas, an antibody against p300 was used and showed that the bands marked in Fig. 4 was p300 (lane 1). Figure 4 Direct physical interaction of Bel1/Tas with p300 determined by co-immunoprecipitation. Bel1/Tas binds to p300 in vitro and in vivo. After transfection of 293T cells with expression plasmids pbel1s and p300FL, cells were labeled with [35S]-Methionine plus [35S]-Cysteine. Cellular extracts were precipitated, separated by SDS-PAGE and exposed. Protein p300FL is marked by bold arrowhead (lanes 1, 3, and 6). In the control, pbel1s was omitted (lane 4). Arrowheads indicate the following protein size markers (M, lanes 2 and 5): myosin of apparent molecular mass of 236, phosphorylase b of 97, and BSA of 66 kDa. To check the data obtained, co-immunoprecipitations were performed with non-labeled 293T cells followed by Western blot analysis. Cellular lysates were prepared from 293T cells separately cotransfected with pbel1s and each one of the three truncated p300 expression plasmids, p300N, p300M, or p300C and analyzed as described above. A polyclonal antibody directed against the Bel1/Tas protein was used in the co-immunoprecipitation followed by immunoblotting with a monoclonal antibody directed against the FLAG epitope fused in-frame with the N-terminus of the three truncated p300 versions. The results shown in Figure 5 indicate that p300N protein specifically interacted with Bel1/Tas (lane 1) whereas the middle and C-terminal regions, p300M and p300C, respectively, do not seem to bind to Bel1/Tas under the conditions used (Fig. 5, lanes 3 and 5). This experiment was repeated several times and yielded the same result. In the controls, pbel1s was omitted in the cotransfections (lanes 2, 4, and 6). Figure 5 Analysis of physical interaction of Bel1/Tas with truncated forms of p300. After cotransfection of 293T cells with 2 μg pbel1s, and, separately with 2 μg each of p300N, p300M, and p300C expression plasmids, cellular extracts were prepared in parallel, divided into equal parts, and analyzed by co-immunoprecipitation and Western blotting. Lysates were precipitated with anti Bel1/Tas antibody and the Western blot was developed with a monoclonal anti-FLAG antiserum. The three arrows in the middle panel mark the correct sizes of the p300N, p300M, and p300C proteins (two blots pasted together). Controls for each p300 plasmid without pbel1s are in lanes 2, 4, and 6. Lower part confirms that the p300N protein specifically binds the Bel1/Tas protein (lane1, marked by arrow); two blots pasted together. Mapping of the p300-Bel1/Tas interaction domain We next determined which N-terminal p300 domain was responsible for the specific interaction with the Bel1/Tas protein. Different truncated versions of p300N (Fig. 1) were prepared, cloned, and subjected to separate immunoprecipitations and Western blot analyses as described above for p300N. In addition, two different GST fusion proteins that contained either the C/H1 or KIX domain were bacterially expressed, purified, and analyzed (Fig. 1). Two different expression plasmids p300N-C/H1-ΔKIX that lacked the KIX but still expressed the C/H1 domain were capable of binding Bel1/Tas (Fig. 6, right panel, lanes 2 and 3). In contrast, the results shown in Fig. 6, lanes 4 and 5, revealed that the plasmid p300-ΔC/H1-ΔKIX that lacks both the C/H1 and KIX domains but retains the short N-terminal region of 196 amino acids did not bind Bel1/Tas. The expression levels of the five constructs were monitored by immunoblot analysis and exhibited the expected bands of p300-derived proteins (Fig. 6, left panel). Figure 6 Mapping of the p300 Bel1/Tas-interaction domain. The p300 expression plasmids, constructs no. 2, and 5 to 8 shown in Fig. 1 were analyzed by immunoprecipitation as described above for p300N. The right hand panel shows the results of immunoblotting obtained after reaction with the monoclonal antibody against the FLAG epitope; arrows and asterisks mark the Bel1/Tas-interacting protein bands from cellular extracts of pbel1s-cotransfected cells (lanes 1–3); intentionally overexposed to visualize the marked bands in lanes 1 and 2. The left panel presents the Western blots of the five recombinant p300N plasmids used for separate co-immunoprecipitations. Numbers in brackets refer to Fig 1. To unambiguously demonstrate that the C/H1 domain of p300 is the Bel1/Tas-interacting region, we performed pull-down assays with two pGST-C/H1 and pGST-ΔC/H1-KIX fusion proteins (Fig. 1, constructs no. 9 and 10). The results revealed that purified pGST-C/H1 clearly interacted with Bel1/Tas as shown in Fig. 7, lane 2 whereas the pGST-ΔC/H1-KIX and a control GST plasmid did not (lanes 1 and 3). Figure 7 Bel1/Tas interacts with GST-C/H1 fusion proteins detected by GST pull-down assays. The recombinant GST-C/H1 and GST-ΔC/H1-KIX proteins were expressed in E. coli BL21 cells and purified by binding to glutathione Sepharose 4B. Each GST-fusion protein bound to glutathione Sepharose 4B was separately mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation and extensive washing with the binding buffer, bound proteins were eluted, separated by SDS-PAGE, and visualized by staining (upper panel); immunoblotting was carried out with an antibody against Bel1/Tas protein (lower panel). To summarize this part, our data show that p300 physically interacted with Bel1/Tas in vivo, and that the C/H1 domain of p300 was responsible for this interaction at least in vitro. Effect of the Bel1-C/H1 domain on Bel1/Tas-mediated activation by p300C To gain more insight into the mechanism of the C/H1-Bel1 complex that affects3 p300C-mediated activation, cotransfections of pbel1s and p300C with expression plasmid p300N-C/H1-ΔKIX were carried out (Fig. 1, construct 6). Cellular lysates of 293T cells were prepared and luciferase assays performed. Cotransfections of fixed concentrations of the pbel1s, 0.5 μg, with the pC/H1-ΔKIX expression plasmid did not enhance p300C-mediated trans-activation (Fig. 8, lanes 5, 7, 9, 11, and 13). In contrast, the expression of the C/H1 domain resulted in a partial suppression of the p300C-driven activation at higher C/H1 domain concentrations (Fig. 8). Similar degrees of inhibition were obtained when lower Bel1/Tas concentrations were used. Western blot analysis was carried out in parallel with increasing concentrations of the coactivator to ascertain Bel1/Tas expression (data not shown). The inhibition by the C/H1 domain explains why the level of trans-activation of p300N and p300FL did not reach the full extent of p300C-driven activation. Figure 8 Effect of p300-C/H1 domain expression on enhancement of p300C-mediated trans-activation. Transient expression assays with pGL3-luc containing the internal BRE promoter after cotransfection of the p300C and pC/H1 expression plasmids with pbel1s. PCAF interacts with Bel1/Tas We next analyzed whether different HAT-expressing genes such as GCN5, PCAF, and SRC-1 were able to enhance and interact with Bel1/Tas. Transient luciferase gene assays with pGCN5 expression plasmids did not affect Bel1/Tas-mediated activation (data not shown). In contrast, when the HAT PCAF expression plasmid was used for co-expression, an enhancement of Bel1/Tas-induced activation was detectable at a concentration of 0.02 μg PCAF DNA and 0.5 μg pbel1s (Fig. 9, upper panel). At higher PCAF DNA concentrations, repression of Bel1/Tas-mediated activation was observed. When the levels of PCAF and Bel1/Tas protein expression was determined by Western blot analysis, a decreased level of the Bel1/Tas expression was detected that was likely due to degradation (Fig. 9, lower panel). The band of the PCAF protein corresponded to the correct size of about 95 kDa (marked by arrow). Figure 9 Functional and physical interaction of Bel1/Tas with PCAF. A, Transient expression assays with pGL3-luc containing the internal BRE promoter after cotransfection of the pCI-FLAG-PCAF with 0.5 μg pbel1s expression plasmid (upper panel). Aliquots were subjected to Western blot analysis (lower panel) We next analyzed the potential interaction between the Bel1/Tas and PCAF proteins by carrying out co-immunoprecipitation with 293T cellular lysates that had been cotransfected with 2.0 μg pbel1s and pCI-PCAF expression plasmids, followed by immunoreaction with an antibody against Bel1 and subsequent immunoblotting with an anti-FLAG antibody to detect PCAF. The results showed that PCAF did indeed interact with the PFV Bel1/Tas activator (Fig. 10, lane 2). As control, an immunoprecipitation and Western blot analysis was performed with the PCAF plasmid in the absence of pbel1s (Fig. 10, lane 1). The result showed that a PCAF band was not detectable under the conditions used. Figure 10 Physical interaction of PCAF with Bel1/Tas. Co-immunoprecipitation and immunoblot analysis of pCI-FLAG-PCAF-cotransfected 293T cells with pbel1s DNA (lane 2). The cellular lysates were incubated with an anti Bel1/Tas antibody and treated as described in the legend to Fig. 5 except for that an antibody directed against the FLAG epitope of PCAF was used for immunoblotting. In the control, cotransfection was done without pbel1s (lane 1). To determine if an additional HAT protein, SRC-1, affected Bel1/Tas-mediated activation, transient reporter gene expression assays were carried out after cotransfection of 293T cells with the coactivator SRC-1a and 0.5 μg pbel1s. Surprisingly, a relatively strong enhancement of Bel1/Tas-mediated activation was detected (Fig. 11, upper panel). The level of expression of both SRC-1a and Bel1/Tas proteins was determined and found to be approximately proportional to the input (Fig. 11, lower panel). Figure 11 Coactivator SRC-1 enhances Bel1/Tas-mediated trans-activation. Reporter luc gene expression assays with pGL3-luc plasmids containing the internal PFV BRE promoter after cotransfection of pCR3.1-FLAG-SRC-1a with the pbel1s expression plasmid (upper panel). Immunoblotting of 293T cellular extracts cotransfected with the SRC-1a and pbel1s expression plasmids (lower panel). To assess whether SRC-1a was able to physically interact with the Bel1/Tas protein, immunoprecipitations and Western blot analysis were carried out under different conditions. The specificity of SRC-1a was ascertained by using monoclonal antibody directed against SRC-1a in control reaction. However, evidence for an interaction between the SRC-1a protein and Bel1/Tas was not obtained. Discussion It was previously reported that Bel1/Tas is capable of inducing the expression of many cellular genes [23]. While it is known that Bel1/Tas binds directly and to a large number of DNA target sites [18-21], the mechanism of activation and the identity of the cellular partner molecules of Bel1/Tas remained unknown. As a first step, we have sought to identify the cellular proteins that interact with the PFV retroviral trans-activator and mediate its activating potential. The data presented here show that the coactivators p300 and HAT PCAF physically bound Bel1/Tas in vitro and both enhanced Bel1/Tas-mediated activation whereas SRC-1 enhanced with Bel1/Tas activation without direct binding. According to our data, Bel1/Tas specifically interacted with the C/H1 domain of p300, although we cannot rule out binding to other p300 domains with much lower affinity not detectable under the rather harsh conditions of co-immunoprecipitation used here. When the levels of the relative luciferase activity of p300FL and its three shortened versions are compared, it is noteworthy that p300C reached the highest level of enhancement of Bel1/Tas-mediated activation (Fig. 2). Besides its intrinsic HAT activity, p300C contains both the intact C/H3 and Q domains that interact with the HAT enzymes PCAF and SRC-1, respectively [10,24,25]. These three HAT enzymes are likely responsible for the large enhancement of the observed trans-activation either directly by acetylation of Bel1/Tas or indirectly by histone acetylation, or both. In contrast to p300C, p300N and p300M do not possess any HAT activities nor do they bind to HAT-containing interaction partners. On the other hand, it is well known that p300 and its three subregions bind a plethora of various partner molecules leading to either activation or repression of transcription. Since the high level of p300C-mediated activation was partially inhibited in cotransfections with C/H1 and pbel1s, competition for Bel1/Tas between the C/H1 and the p300C-terminal interaction partners PCAF and SRC-1 cannot be ruled out so that both direct and indirect mechanisms might be responsible for the relative increase in Bel1/Tas-mediated activation by p300C. When Bel1/Tas binds to the C/H1 domain, the degree of Bel1/Tas acetylation may be much lower, since the protein surface of Bel1/Tas may be occluded and, hence less accessible. Some residual Bel1 acetylation might still occur by endogenous p300 and PCAF. Other factors might play additional roles. The ability of full-length p300 to trans-activate Bel1/Tas was relatively low for two reasons. First, the transfection efficiency of the full-length p300 is very low because of its large plasmid size, and the concentration of endogenous p300 is limiting. Secondly, the activation loop of p300 HAT is not fully activated by auto-acetylation as required for full trans-activation [26]. Since the relative activation by SRC-1 was not as high as that of p300C, we consider the HAT activity of PCAF as one of the major players of Bel1/Tas-mediated trans-activation. This result is supported by the observed enhancement of Bel1/Tas activation after cotransfection with PCAF that resulted in higher levels of Bel1 acetylation thereby leading to increased binding to the IP.BRE [J. Bodem, personal communication]. Thus, the observed high level of enhancement of p300C might be due to the synergistic effects brought about by formation of ternary p300C-PCAF-Bel1 and binary p300C-SRC complexes, respectively. In these multimeric protein complexes, Bel1/Tas binds p300C indirectly through PCAF. Consistent with the HAT activities of PCAF and p300, we detected acetylated Bel1/Tas in pbel1s-transfected 293T cells using monoclonal antibody against acetyl-Lysine after immunoblotting (our unpublished data). It is intriguing that the distribution of the closely spaced Lysines of Bel1/Tas apparently mimics the correspondingly spaced Lysines in histones. This observation is further complicated by our observation that cotransfection with higher levels of PCAF led to a reduced stability of Bel1/Tas (Fig. 9). We assume that PCAF acetylates or even hyper-acetylates the Bel1/Tas protein at closely spaced Lysines in analogy to other activators reported previously [9]. The decreased stability of acetylated Bel1/Tas might indicate that modified Bel1/Tas is less stable than the unmodified form. This observation adds an additional layer of combinatorial regulation to Bel1/Tas-mediated trans-activation. It is intriguing that some viral trans-activators interact with more than a single p300 domain [10,14]. However, Bel1/Tas might recruit a second interacting region of p300 through binding PCAF (Fig. 10) that is known to interact with a p300 domain different from the C/H1 domain (Fig. 1) [10]. The complex nature of p300-Bel1/Tas interactions reported here might serve to strengthen the overall binding affinity between Bel1/Tas and the PCAF interaction domain of p300 within a larger transcriptional complex [27]. PCAF is known to specifically acetylate distinct Lysine residues of a subset of core histones and thereby regulate the transcriptional activity of many genes depending on the genetic context. It is well documented that acetylation, methylation and other covalent histone modifications are essential signals for the regulation of transcription [28]. It remains to be seen whether the stronger level of enhancement of p300C is a special if not unique feature of Bel1/Tas activation and due to over-expression or to repressive effects of other p300-interacting protein factors that cannot bind to the truncated p300 protein. Alternatively, many other factors were reported to bind to the C-terminal domains of p300 that also encompass general transcription factors TBP and TFIIB proteins that might also be responsible for the enhancement observed here [1-3]. In search of viral and cellular activators that are comparable with the ability of Bel1/Tas to interact with the C/H1 domain of p300, we found one case. A report indicates that EBNA-2 protein shares many features with Bel1/Tas that include the C-terminal acidic activation domain as well as the abilities to bind both the C/H1 domain and PCAF [12]. There remain two differences, however. First, EBNA-2 binds to both the C/H1 and the C/H3 domain, and, secondly, PCAF does not coactivate EBNA-2 in strong contrast to Bel1/Tas [12]. Of note, an additional HAT enzyme, GCN5, did not interact with Bel1/Tas when tested in reporter genes assays indicating that only distinct HAT sets such as those identified in this report specifically interact with Bel1/Tas in trans-activation. The precise roles of the HAT activities of PCAF, p300, and SRC-1 during Bel1/Tas-mediated trans-activation remain to be addressed in future studies. Conclusions Coactivators PCAF and p300 were identified to physically and functionally interact with the spumaviral Bel1/Tas trans-activator. Coactivator SRC-1 was found to strongly enhance Bel1/Tas-mediated trans-activation. The C/H1 domain of p300 was responsible for binding the retroviral activator and found to partially inhibit the p300-driven trans-activation. Methods Antibodies Mouse monoclonal antibodies directed against the FLAG epitopes of p300FL, p300N, p300M, p300C, and PCAF were purchased from Sigma, rabbit polyclonal antibodies against the N-terminal p300FL, the C-terminal p300C, and SRC-1a from Santa Cruz Biotechnology. The polyclonal serum direct against Bel1/Tas was used as described previously [23]. Typically, 5 μl of each antiserum was used for each immunoprecipitation. Monoclonal antibody against acetyl-Lysine was purchased from Sigma. Plasmids, cells, transfections, and metabolic labeling Plasmids pUC18, pCMVβ-gal, pbel1s [29], pGL3-pro-IP. BRE (-1 to -192), pCI-FLAG-p300FL, pCI-FLAG-p300N, pCI-FLAG-p300M, pCI-FLAG-p300C [4], and pCI-FLAG-PCAF [29] were separately, or in the combinations indicated, transfected into 293T cells using Lipofectamine 2000 (Invitrogen). In general, unless otherwise indicated, 0.1 – 10 μg plasmid DNA were transfected into 293T cells grown in Petri dishes with a diameter of six cm. Full-length pCI-FLAG-300FL plasmids and three different shortened versions (Fig. 1, constructs no. 1 to 4) were grown in E. coli, DH5α cells [4]. The PFV internal promoter was constructed by PCR-mediated amplification of the defined promoter fragments as reported previously [19,21]. Recombinant clone p300N-C/H1-ΔKIX* (construct no. 5, Fig. 1), was constructed by first generating two PCR products with pCI-p300FL as template using the sense (s) and antisense (as) primers s1: 5'-CTTATGGTTCACCATATACTCAGAATCC-3', as1: AAACTGGAACCATGCCTGCATTTCTCTTATCACC-3', s2: 5'-GAAATGCAGGCATGGTTCCAGTTTCCAT-3', and as2: 5'-GGAAGGAACTGGCCCTGGTTGGAAGGCTGTTG-3' to amplify the sequence from nucleotide 755 to 1275 and nucleotides 1984 to 2297 fused in-frame. The resulting DNA product of 833 nucleotides was cloned into the pCR2.1 Topo vector and designated as pCR2.1-ΔKIX. An SphI-NotI DNA fragment obtained from pCR2.1-ΔKIX and was inserted into pCI-p300N that had been predigested with SphI and NotI. The borders of construct no. 5 are shown in Fig. 1 and the expressed recombinant protein had the expected size. p300-C/H1-ΔKIX (no. 6) was constructed by inserting the SphI/NotI DNA fragment from pGEX-C/H1 (35) into pCI-p300N digested with SphI/NotI for expression of residues 1 to 424 of p300. Construct no. 7 (Fig. 1) was constructed by digesting pCI-p300N with SphI and re-ligating the larger fragment for the expression of residues 1 through 347 of p300. Finally, p300ΔN-ΔC/H1-ΔKIX was constructed from pCI-p300N by digesting with MunI and re-ligated to express residues 1 to 196 of p300. Bacterial plasmids coding for glutathion-S-transferase (GST) fusion proteins pGST-C/H1 (328–424) and pGST-KIX (436–661) (Fig. 1) were constructed from pGEX-6p-2GST-p300 [30] and grown in E. coli, BL21 cells. pCR3.1-FLAG-SRC-1a was grown in E. coli, JM109 cells [24]. Human 293T or HeLa cells were cultivated in DMEM medium supplemented with 1% penicillin and streptomycin, 1% Glutamine and 10 % fetal calf serum. Plasmid p300FL was transfected into 293T cells and metabolically labeled with L-[35S]-Methionine plus L-[35S]-Cysteine (spec. act. of 37 TBq/mM, PRO-MIX, Amersham) for 6 hr. Cells were harvested and used for immunoprecipitation as described above. The precipitates were analyzed by SDS-PAGE on 12% gels, dried, and exposed on KODAK Biomax MR1 films. Luc reporter gene expression assays Plasmid pCMV-βgal directing β-galactosidase expression from the CMV-IE promoter was used for normalization of transfection efficiency. Luc reporter gene assays were performed and quantified as described [31] using a Luminoskan TL Plus luminometer (Labsystems, Frankfurt, FRG). pUC18 vector plasmid DNA was used as carrier DNA to equalize the DNA concentration of each transfection. Cells were harvested 18 h after transfection. The results of luc assays were based upon at least triplicate experiments on multiple independent occasions. Expression levels were monitored by Western blot analysis. Co-immunoprecipitation Immunoprecipitation was performed as described previously with minor modifications [32]. Lysates of subconfluent cotransfected layer of 293T cells were prepared by first washing the cells with PBS, and subsequently lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], 1 mM phenylmethylsulfonyl-fluoride containing 1% (v/v) Triton X-100. To inhibit unspecific protease activity, protease inhibitors (Biomol) were added to the lysis buffer. Lysates were precleared with protein A-SepharoseCL-4B (Amersham Bioscience AB, Uppsala). Co-immunoprecipitation of p300FL, p300N, p300M, p300C, SRC-1, and PCAF were performed with rabbit anti Bel1 antiserum [23]. The immune precipitates were retrieved with protein A-SepharoseCL-4B (Pharmacia) and eluted by boiling. To detect the specific precipitate, immunoblotting was performed with specific antibodies against expressed p300, shortened p300 versions, and SRC-1. For the detection of expressed PCAF, Western blot analysis with monoclonal anti-FLAG antibody was carried out. The immunoprecipitates were washed three times with the lysis buffer and analyzed by immunoblotting on 12% gels for expressed p300FL and on 14% gels for shortened p300 forms, PCAF, and SRC-1a. The experiments were repeated at least three times, specially the immunoprecipitations of lysates of p300C-transfected cells. GST pull-down assays The recombinant GST-C/H1 and GST-KIX proteins were expressed in E. coli BL21/DE3 cells after transformation with the corresponding plasmids. Expressed proteins were purified by binding to glutathione Sepharose 4B resin. Each of the GST-fusion proteins bound to glutathione Sepharose 4B were mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation at 4°C overnight in binding buffer [30] and extensive washing with the binding buffer, bound proteins were eluted, separated by SDS-PAGE, and visualized by staining with Coomassie Brilliant Blue according to Ariumi [30] or detected by Western blot analysis. Immunoblotting Cells were harvested two days after transfection by lysis in 1% SDS and the protein concentration was determined using the DC protein assay (BioRad). Identical amounts of proteins were separated by SDS-PAGE on 12% gels, blotted, reacted with monoclonal serum directed against the FLAG epitope of the four different FLAG-tagged p300 (Sigma), or polyclonal serum direct against Bel1 [23], and detected by enhanced chemoluminescence. List of abbreviations used CBP, CREB-binding protein; C/H1, 2, and 3, Cys/His-rich domains of p300; FL, full-length; HAT, histone acetyltransferase; LTR, long terminal repeat; luc, luciferase; PCAF, p300/CBP-associated factor; Q, Glutamine-rich domain of p300; SRC, steroid receptor coactivator; p300N, p300M, and p300C, amino-, middle and C-terminal regions of p300; PFV, primate foamy virus; IP.BRE, internal promoter Bel1 response element; TAD, trans-activation domain; NF1, nuclear factor 1; CMV, cytomegalovirus; GST, glutathione-S-transferase. Authors' contributions HB and WM contributed equally to the manuscript. VO carried out the molecular cloning of p300 derivatives. YN participated in the design of the study. All authors read and approved the final manuscript. Acknowledgments We thank Kunitada Shimotohno for critically reading the manuscript and helpful comments and our colleagues for generously providing plasmids and reagents: Shelley Berger, Don Chen, Ella Englander, Richard Gronostajski, Tasuku Honjo, Steve McMahon, Bert O'Malley, Marc Montminy, Sharon Roth, and Kunitada Shimotohno. We are grateful to Jennifer Reed for critically reading our paper and to Gholamreza Darai and Harald zur Hausen for support. The Fond der Chemischen Industrie supported this work. ==== Refs Goodman RH Smolik S CBP/p300 in cell growth, transformation, and development Genes Dev 2000 14 1553 1577 10887150 Vo N Goodman RH CREB-binding protein and p300 in transcriptional regulation J Biol Chem 2001 276 13505 13508 11279224 Shikama N Lyon J La Thangue NB The p300/CBP family: integrating signals with transcription factors and chromatin Trends Cell Biol 1997 7 230 236 17708951 10.1016/S0962-8924(97)01048-9 Ogryzko V Schiltz RL Russanova V Howard BH Nakatani Y The transcriptional coactivators p300 and CBP are histone acetyltransferases Cell 1996 87 953 959 8945521 10.1016/S0092-8674(00)82001-2 Grossman SR Deato ME Brignone C Chan HM Kung AL Tagami H Nakatani Y Livingston DM Polyubiquitination of p53 by a ubiquitin ligase activity of p300 Science 2003 300 342 344 12690203 10.1126/science.1080386 Hottiger MO Nabel GJ Viral replication and the coactivators p300 and CBP Trends Microbiol 2000 8 560 565 11115752 10.1016/S0966-842X(00)01874-6 Caron C Col E Khochbin S The viral control of cellular acetylation signaling Bioessays 2003 25 58 65 12508283 10.1002/bies.10202 Featherstone M Coactivators in transcription initiation: here are your orders Curr Opinion Gen & Develop 2002 12 149 155 10.1016/S0959-437X(02)00280-0 Gu W Roeder RG Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain Cell 1997 90 595 606 9288740 10.1016/S0092-8674(00)80521-8 Yang X-J Ogryzko V Nishikawa J Howard BH Nakatani Y A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A Nature 1996 382 319 324 8684459 10.1038/382319a0 Connor MJ Zimmermann H Nielsen S Bernard H-U Kouzarides T Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif, (TRAM) in CBP/p300 J Virol 1999 73 3574 3581 10196247 Wang L Grossman S Kieff E Epstein-Barr virus nuclear protein 2 interacts with p300, CBP, and PCAF histone acetyltransferases in activation of the LMP1 promoter Proc Natl Acad Sci 2000 97 430 435 10618435 10.1073/pnas.97.1.430 Kwok RPS Lawrance ME Lundblad JR Goodman PS Shih H-M Connor LM Marriott SJ Goodman RH Control of cAMP-regulated enhancers by the trans-activator Tax through CREB and the co-activator CBP Nature 1996 380 642 646 8602268 10.1038/380642a0 Georges SA Giebler HA Cole PA Luger K Laybourn PJ Nyborg JK Tax recruitment of CBP/p300 via the KIX domain, reveals a potent requirement for acetyltransferase activity that is chromatin dependent and histone tail independent Mol Cell Biol 2003 23 3392 3404 12724399 10.1128/MCB.23.10.3392-3404.2003 Schiltz RL Nakatani Y The PCAF acetylase complex as a potential tumor suppressor Biochim Biophys Acta 2000 1470 M37 M53 10722926 10.1016/S0304-419X(99)00037-2 Leo C Chen JD The SRC family of nuclear receptor coactivators Gene 2000 245 1 11 10713439 10.1016/S0378-1119(00)00024-X Josephson NC Vassilopoulos G Trobridge GD Priestley GV Wood BL Papayannopoulou T Russell DW Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors Proc Natl Acad Sci 2002 99 8295 8300 12060773 10.1073/pnas.122131099 He F Blair WS Fukushima J Cullen BR The human foamy virus Bel1 transcription factor is a sequence-specific DNA binding protein J Virol 1996 70 3902 3908 8648727 Kido K Doerks A Löchelt M Flügel RM Identification and functional characterization of an intragenic DNA binding site for the spumaretroviral trans-activator in the human p57KIP2 gene J Biol Chem 2002 277 12032 12039 11815601 10.1074/jbc.M108747200 Löchelt M Muranyi W Flügel RM Human foamy virus possesses an internal, bel-1-dependent and functional promoter Proc Natl Acad Sci USA 1993 90 7217 7321 Kido K Bannert H Gronostajski RM Flügel RM Bel1-mediated repression of the spumaretroviral internal promoter is repressed by nuclear factor I J Biol Chem 2003 278 11836 11842 12446690 10.1074/jbc.M208963200 Chaudhry AZ Vitullo AD Gronostajski RM Nuclear factor I-mediated repression of the mouse mammary tumor virus promoter is abrogated by the coactivators p300/CBP and SRC-1 J Biol Chem 1999 274 7072 7081 10066764 10.1074/jbc.274.11.7072 Wagner A Doerks A Aboud M Alonso A Tokino T Flügel RM Löchelt M Induction of cellular genes is mediated by the Bel1 trans-activator in foamy virus-infected human cells J Virol 2000 74 4441 4447 10775579 10.1128/JVI.74.10.4441-4447.2000 Yao T-P Ku G Zhou N Scully R Livingston D The nuclear hormone receptor coactivator SRC-1 is a specific target of p300 Proc Natl Acad Sci 1996 93 10626 10631 8855229 10.1073/pnas.93.20.10626 Chen H Lin RJ Schiltz RL Chakravarti D Nash A Nagy L Privalky ML Nakatani Y Evans RM Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with PCAF and CBP/p300 Cell 1997 90 569 580 9267036 10.1016/S0092-8674(00)80516-4 Thompson PR Wang D Wang L Fulco M Pediconi N Zhang D An W Ge Q Roeder RG Wong J Levrero M Sartorelli V Cotter RJ Cole PA Regulation of the p300 HAT domain via a novel activation loop Nature Struc & Mol Biol 2004 11 308 315 10.1038/nsmb740 Ogryzko V Kotani T Zhang X Schiltz RL Howard T Yang X-J Howard BH Qin J Nakatani Y Histone-like TAFs within the PCAF histone acetylase complex Cell 1998 94 35 44 9674425 10.1016/S0092-8674(00)81219-2 Strahl BD Allis D The language of covalent histone modifications Nature 2000 403 41 45 10638745 10.1038/47412 Venkatesh LK Yang C Theodorakis PA Chinnadurai G Functional dissection of the human spumaretrovirus transactivator identifies distinct classes of dominant-negative mutants J Virol 1993 67 161 169 8380068 Ariumi Y Kaida A Lin J-Y Hirota M Masui O Yamaoka S Taya Y Shimotohno K HTLV-1 Tax oncoprtein represses the p53-mediated trans-activation through coactivator CBP sequestration Oncogene 2000 19 1491 1499 10734308 10.1038/sj.onc.1203450 Schreiber E Matthias P Müller MM Schaffner W Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells Nucleic Acids Research 1989 17 6419 6420 2771659 Ziegler H Muranyi W Burgert H-G Kremmer E Koszinowski UH The luminal part of the murine cytomegalovirus glycoprotein gp40 catalyzes the retention of MHC class I molecules EMBO J 2000 19 870 881 10698929 10.1093/emboj/19.5.870

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==== Front BMC PharmacolBMC Pharmacology1471-2210BioMed Central London 1471-2210-4-181533102010.1186/1471-2210-4-18Research ArticleEffect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans Tsuneki Hiroshi [email protected] Mitsuyo [email protected] Miki [email protected] Jin-Bin [email protected] Toshiyasu [email protected] Ikuko [email protected] Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan2 Toyama College, Toyama 930-0193, Japan3 China Medical College, Taichung, Taiwan Republic of China2004 26 8 2004 4 18 18 4 6 2004 26 8 2004 Copyright © 2004 Tsuneki et al; licensee BioMed Central Ltd.2004Tsuneki et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Green tea is widely consumed in Asian countries and is becoming increasingly popular in Western countries. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The present study was aimed at providing evidence of improvement in glucose metabolism in diabetic mice and healthy humans upon green tea consumption. Results Green tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body in oral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ mice and streptozotocin-diabetic mice 2–6 h after administration at 300 mg/kg without affecting serum insulin level, whereas no effect was observed in control mice (+m/+m and normal ddY mice). The serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI (surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), and then compared to investigate any effects of oral green tea administration on serum proteins. The protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios (m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075, 17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peak intensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peak intensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after the administration. Conclusion The present study provides evidence that green tea has an antidiabetic effect. Although we could not find simple reversed effect of green tea on the diabetes-induced modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein level that was decreased in the diabetic state was further decreased after green tea administration. This is the first report demonstrating that a certain serum protein may be involved in the antihyperglycemic effect of green tea. The contribution of this protein should be further studied. ==== Body Background Green tea (leaves of Camellia sinensis, Theaceae) is a popular beverage in East Asia, and also used as a herbal remedy in Europe and North America. Green tea is considered to be antiinflammatory, antioxidative, antimutagenic, and anticarcinogenic [1,2], and can prevent cardiac disorders. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The amelioration of insulin resistance by green tea is associated with the increased expression level of glucose transporter IV in a fructose-fed rat [3]. Green tea extract contains polyphenols (e.g., catechin, epicatechin, epigallocatechin, and their gallates), teanin and caffeine. The extract also includes pyrroloquinoline quinone, a newly discovered vitamin [4]. Some constituent components have been shown to enhance the basal and insulin-stimulated glucose uptake of rat adipocytes [5], to inhibit intestinal glucose uptake by inhibiting the sodium-dependent glucose transporter of rabbit intestinal epithelial cells [6], and to reduce serum glucose level in alloxan-diabetic rats [7]. Controversially, caffeine acutely lowers insulin sensitivity in humans [8]. The present study was aimed at providing evidence of the improvement in glucose metabolism in humans and diabetic mice upon green tea consumption. Furthermore, to investigate whether some serum proteins mediate the effects of green tea on hyperglycemia in the diabetic state, proteomic analyses were performed using SELDI (surface enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), because the SELDI-TOF-MS enables the measurement of the relative abundance of proteins of various sizes in a blood sample. Here, we compared the proteomic patterns of sera from diabetic db+/db+ and wild-type mice, and further investigated the influence of green tea administration on the proteomic patterns of serum in diabetic mice. If the diabetic modifications of serum protein levels are reversed by green tea administration, these affected proteins can be used as therapeutic targets in diabetes. Results Oral glucose tolerance test (OGTT) We investigated whether oral glucose tolerance is improved in healthy volunteers by drinking a suspension of green tea powder. Blood glucose levels were measured before and 30, 60, and 120 min after drinking 1.5 g of green tea. The effects of hot water without tea powder on blood glucose level in the same participants were also investigated. Fig. 1 shows that glucose tolerance was substantially improved with tea administration compared with hot water administration. In detail, glucose metabolism increased in 14 participants, remained unchanged in 3 participants, and worsened in 5 participants. Basal blood glucose levels (BGLs) did not significantly change in all participants (Fig. 1). Figure 1 Increase in glucose metabolism in healthy humans administered with green tea. Oral glucose tolerance curves (OGTT, n = 22) and basal blood glucose levels (Basal, n = 25) before and 30, 60 and 120 min after administration with either a suspension of green tea powder (●) or hot water (○). All participants were fasted since the last supper before starting the experiment at 9:30 a.m. In the glucose tolerance test, 225 ml of Trelan-G75 containing 75 g glucose was perorally administered 10 min after drinking a suspension of green tea powder (1.5 g/150 ml hot water) or hot water (150 ml). Effect of green tea or hot water on basal blood glucose levels was examined without administrating Trelan-G75. Blood glucose levels (mg/dl) measured were averaged in each treatment group, and compared by cross-over test. Values represent the means ± S.E.M. *P < 0.05, **P < 0.01; significantly different from the vehicle control at each time-point, determined by one-way ANOVA, followed by Scheffé's test. Antihyperglycemic effects of green tea in diabetic mice The crude extract of fresh tea leaves picked from either No.12 or No. 13 cultivar at 300 mg/kg significantly lowered BGL 2–6 h after peroral administration in streptozotocin (STZ)-diabetic ddY mice in the fasting state (Fig. 2). In the saline-treated control group (n = 5), BGL slightly changed from 273 ± 28 mg/dl to 241 ± 28 mg/dl after 6 h, whereas in the green-tea (No. 12)-treated group (n = 3), BGL markedly changed from 235 ± 15 mg/dl to 116 ± 12 mg/dl. Similarly, BGL markedly changed from 201 ± 10 mg/dl to106 ± 8 mg/dl after 6 h in the green-tea (No. 13)-treated group (n = 3). Figure 2 Antihyperglycemic effects of green tea leaf extracts in the fasting STZ-diabetic ddY mice. The dried extracts of fresh tea leaves prepared at the Taiwan Tea Experiment Station [TTES, Cultivar No. 12 (Chinhsuan) and No.13 (Tzuiyu)] were dissolved in saline and perorally administered at 300 mg/kg into STZ-diabetic ddY mice in the fasting state. Blood glucose levels 2 h, 4 h, and 6 h after administration of green tea leaf extracts are expressed as percentages of the glucose levels at 0 h (before the administration) in each mouse, and then averaged. Values represent the means ± S.E.M. (n = 3–5). *P < 0.05; significantly different from the blood glucose levels of STZ-diabetic mice administered with saline (control) at each time-point, determined by one-way ANOVA, followed by Scheffé's test. The antihyperglycemic effects of green tea powder suspension at 30, 150, and 300 mg/kg were examined 2 h after administration to the STZ-diabetic mice in the fasting state. As shown in Fig. 3, green tea tended to lower BGL at 150 mg/kg, and significantly lowered BGL at 300 mg/kg. Furthermore, the effect of green tea was compared among different groups of mice, i.e., STZ-diabetic ddY mice, normal ddY mice, diabetic db+/db+ mice and +m/+m mice (Fig. 4). BGLs were significantly lowered in both STZ-diabetic mice and db+/db+ mice 2 h after administration of green tea powder suspension (300 mg/kg), whereas no significant changes were observed in normal ddY mice and +m/+m mice. Figure 3 Antihyperglycemic effects of green tea powder suspension in the fasting STZ-diabetic ddY mice. STZ-diabetic ddY mice were perorally administered with a suspension of green tea powder at 30, 150, and 300 mg/kg in the fasting state. Blood glucose levels 2 h after the administration of green tea are expressed as percentages of the glucose level 0 h before the administration in each mouse, and then averaged. Values represent the means ± S.E.M. (n = 6 – 20). **P < 0.01; significantly different from the blood glucose levels of STZ-diabetic mice administered with saline (control, 0 mg/kg of green tea), by one-way ANOVA, followed by Scheffé's test. Figure 4 Blood-glucose-lowering effects of green tea in diabetic mice but not in normal mice. STZ-diabetic ddY, normal ddY, diabetic db+/db+, and wild-type (+m/+m) mice were perorally administered with either a suspension of green tea powder at 300 mg/kg or saline in the fasting state. Blood glucose levels (mg/dl) were measured before and 2 h after the administration, and then averaged. Values represent the means ± S.E.M. (n = 5 – 21). *P < 0.05, **P < 0.01; significantly different from values at 0 h (before the administration), by one-way ANOVA, followed by Scheffé's test. No elevation of blood insulin levels was observed during the decrease in BGL either by tea leaf extract or by green tea suspension in STZ-diabetic mice (data not shown). These results suggest that both fresh green tea leaf extract and green tea suspension lower BGL without changing serum insulin concentration. Serum protein profiles altered in diabetic db+/db+ mice To explore the mechanisms underlying the antihyperglycemic effect of green tea, proteomic patterns of sera from diabetic (db+/db+) and wild-type (+m/+m) mice were first investigated and compared to identify peaks specific to the diabetic state using SELDI-TOF-MS. Samples of sera from fasting +m/+m mice were loaded onto CM10 (a cationic exchanger, pH4) ProteinChip arrays (Fig. 5). The other types of ProteinChip Array, i.e., CM10 (a cationic exchanger, pH7), Q10 (an anionic exchanger, pH8) and IMAC30-Cu2+ (immobilized metal affinity chromatography, pH7), were also used to fractionate proteins in the serum. Then, these arrays were analyzed using a ProteinChip system, and the amount of individual serum proteins was estimated from the peak intensity of the mass spectral signal and the mass/charge ratio (m/z) equivalent to the molecular weight of each protein. All the spectral peaks of serum proteins from diabetic mice, which were significantly different (P < 0.05, by unpaired t-test) in intensities from those of serum proteins from wild-type mice, are shown in Fig. 6A: the peak intensities of proteins at m/z 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those at m/z 13075, 17406, 17407, 17418, 17622, 18431, 26100 were >3 times higher than respective peak intensities of proteins of sera from +m/+m mice. Typical spectra are shown in Figs. 6B and 6C (see additional file 1 concerning the other peaks). Figure 5 Analysis of proteomic profiles in mouse serum using SELDI-TOF-MS. Serum samples from fasting wild-type (+m/+m) mice were loaded onto the spots of CM10 (a cationic exchanger, pH4) ProteinChip arrays. The spots were analyzed using the SELDI ProteinChip system on three different ranges: m/z 3000 – 10000, m/z 10000 – 20000 and m/z 20000 – 100000. The y-axis of the spectra indicates the mass-to-charge ratio (m/z) of protonated proteins, and the x-axis indicates the relative intensities of mass spectral signals. Note: The intensity of each peak is directly proportional to the amount of protein, but the peak intensities could not be comparable among the different proteins because of the difference in ionization. Figure 6 Proteomic analyses demonstrating the differences in protein profiles of sera from diabetic and wild-type mice. Serum samples from fasting diabetic (db+/db+) mice and fasting wild-type (+m/+m) mice were loaded onto ProteinChip arrays. (A) List of peaks of proteins and/or peptides with indicated m/z values, the peak intensities of which were significantly changed in the diabetic state. Relative peak intensities were averaged (n = 8). The fold changes are presented as ratios of the peak intensities at indicated m/z values in db+/db+ to those in the +m/+m mice. Peaks at m/z 4203, 4576, 8515, 9291, 17406 and 18678 were obtained using CM10 ProteinChip (pH4). Peaks at 8733 and 9311 m/z were obtained using CM10 ProteinChip (pH7). Peaks at m/z 3933, 4119, 4206, 4369, 4566, 4579, 4637, 8523, 8827, 8915, 9283, 13075, 17407, 17418, 17622, 18431, 18691, 22334 and 26100 were obtained using Q10 ProteinChip. Peak at m/z 4211 was obtained using IMAC30. The chips were analyzed by SELDI-TOF-MS. (B) and (C) Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (upper, representative of 4–8 independent observations) and the peak intensity averages at m/z 4206 and 26100 (lower, n = 4 for +m/+m, n = 8 for db+/db+). The analyzed peak is indicated by arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak in wild-type mice, by unpaired t-test. Modification of serum protein profiles of diabetic db+/db+ mice by green tea Serum protein profiles of db+/db+ mice were investigated 2 h after green tea administration, and compared with those administered with saline (control). Again, we confirmed a significant decrease in BGL by green tea in the blood samples used in these proteomic analyses (data not shown). All the protein signals that were significantly changed (P < 0.05, by unpaired t-test) in terms of peak intensity by the green tea administration, but not by the saline administration, are described in Fig. 7A. The serum proteomic patterns of the green-tea-treated group demonstrate that the peak intensities of proteins at m/z 11651 and 11863 decreased to less than one third after 2 h of tea administration, whereas no significant changes were observed in the control group. Typical spectra are shown in Fig. 7B (see additional file 2 for the change at m/z 11651). When the results in Fig. 7A are compared with those in Fig. 6A to identify protein peaks specific for diabetes and which are sensitive to green tea administration, a peak at m/z 4211 (4212) was found (Fig. 8). The intensity of this peak was significantly lowered in the diabetic state (Fig. 8A), and was significantly decreased 2 h after green tea administration (Fig. 8B). Figure 7 Changes in serum protein profiles in db+/db+ mice by administration of green tea. Serum samples from fasting diabetic (db+/db+) mice 2 h after administration of green tea suspension were loaded onto ProteinChip Arrays. The chips were analyzed using SELDI-TOF-MS. (A) List of peaks of proteins and/or peptides with indicated m/z values, the peak intensities of which were significantly changed by the green tea administration. Relative peak intensities were averaged (n = 4). The fold changes are presented as ratios of the peak intensities at indicated m/z values 2 h after to before green tea administration. Peaks at m/z 7495, 7595, 7808, 7920, 14983, 15612 and 15614 were obtained using CM10 ProteinChip (pH4), whereas those at m/z 7503, 7611, 7823, 7926, 11651, 11664, 11863, 15004 and 15638 were obtained using CM10 ProteinChip (pH7). Peaks at m/z 4212, 4226, 7499, 11637, 11846, 13711, 13831, 14974, 15180, 31204 and 65906 were obtained using IMAC30 ProteinChip. (B) Typical data of relative peak intensities (upper, representative of 4 independent observations) and the peak intensity average at m/z 11863 (lower, n = 4) after green tea administration and saline control. The analyzed peak is indicated by arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak obtained before the administration, by unpaired t-test. Figure 8 Changes in peak intensity at m/z 4211 in diabetic state and after green tea administration. (A) Decrease in the peak intensity at m/z 4211 in serum samples from fasting diabetic (db+/db+) mice, compared with that of the fasting wild-type (+m/+m) mice. Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (upper, representative of 4–8 independent observations) and the averaged intensities of the peak at m/z 4211 (lower, n = 4 for +m/+m, n = 8 for db+/db+) indicated above by arrows. **P < 0.01; significantly different from the peak in wild-type mice, by unpaired t-test. (B) Decrease in the peak intensity at m/z 4212 in serum samples from fasting db+/db+ mice 2 h after green tea administration. Typical data of relative peak intensity (upper, representative of 4 independent observations) and the averaged intensities of the peak at m/z 4212 (lower, n = 4) after administration with either green tea or saline. **P < 0.01; significantly different from the peak obtained before the administration, by unpaired t-test. The relative peak intensities of hemoglobin-related multisignals ranging from m/z 14974 to 15638 [9] were increased by green tea administration (Fig. 7A). Parallel changes were observed at half and double these m/z values (Fig. 7A, 9) (see additional file 2 concerning these changes). Since multicharged protein ions are apparently observed as proteins of different sizes in the mass spectrometry, these changes seem to reflect the modification of one group of proteins (hemoglobin-related proteins) by green tea intake. Figure 9 Hemoglobin-related multiple SELDI-TOF-MS signals in db+/db+ mice. Samples of sera from nontreated db+/db+ mice in the fasting state were loaded onto ProteinChip arrays. Left panel: hemoglobin-related multi-MS signals. From the average of 8 data, the signals were detected from m/z 14974 to 15638 (+H) using CM10 ProteinChip (pH7). The double-charged m/z values appear to be observed from m/z 7495 to 7823 (+2H). These values may correspond to hemoglobin α- and β-chains, and other hemoglobin-related proteins. Right panel: multi-MS signals observed at approximately double the single-charged m/z values for the hemoglobin-related signals, using IMAC ProteinChip. These signals may correspond to the dimers of hemoglobin α- and β-chains. Discussion Green tea is widely consumed in Asian countries, while black tea is most popular in Western countries. The manufacturing process of green tea differs from that of black tea because freshly picked young leaves of the tea are immediately steamed. This process destroys the enzymes responsible for breaking down the color pigments in the leaves and allows the tea to maintain its green color during the subsequent rolling and drying processes. The amounts of constituent compounds are slightly different from those of black tea. Pharmacological studies using constituent compounds in green tea have been recently reviewed by Kaszkin et al. [10]. Green tea extracts are more stable than pure epigallocatechin gallate, the major constituents of green tea, because of the presence of other antioxidant constituents in the extract [10]. In general, herbal medicines are complex mixtures of different compounds that often act in a synergistic fashion and exert their full beneficial effect as total extracts [11]. In the present study, we demonstrated that green tea produces an antihyperglycemic effect without affecting insulin secretion in STZ-diabetic mice. We therefore explore the mechanism underlying the green tea effect by investigating the serum protein profiles of db+/db+ mice, a genetic model of type 2 diabetes, using SELDI-TOF-MS. First, we performed a preliminary analysis to determine the peaks (molecular weights) of biomarker proteins that were abnormally contained in the serum of diabetic mice, without identifying individual serum proteins. As a result, we found that the levels of several serum proteins were significantly altered in the diabetic state. Secondly, we investigated which marker proteins are affected by green tea administration. Despite the changes in the levels of several serum proteins after green tea administration, none of the protein peaks specific for diabetes were sensitive to the administration, except for a peak at m/z 4211(4212). The level of this 4211(4212) Da protein was reduced both in the diabetic state and by green tea administration. Thus, no simple reversed effect of green tea on the diabetes-induced modifications of serum protein levels was observed. The 4211(4212) Da protein has not yet been identified, but only two candidate groups of vertebrate proteins are found by scanning Swiss-Prot database (molecular weight, 4211; molecular weight ranges, 0.1% according to the standard errors of the current MS analysis; pI, 6; pI ranges, 10): pancreatic polypeptide (PP) (primary accession numbers P13083, P37999, P41519, P38000, P11967) and antibacterial peptides [β-defensin C7 precursor (018815), 4 kDa defensin (P56686), cryptidine-5 precursor (P28312), antibacterial peptide BMAP-34 precursor (P56425)]. PP is primarily expressed in the endocrine cells of the pancreas, and the plasma PP concentrations are elevated by food intake [12]. Peripheral PP administration results in a reduction in food intake and an increase in energy expenditure [12]. Interestingly, a previous study revealed that PP-containing cell populations in the islets of Langerhans are reduced in db+/db+ mice with mild hyperinsulinemic diabetes [13], which is consistent with the observed reduction in the intensity of the peak at m/z 4211(4212) in the diabetic state. We speculate that the reduction of BGL by green tea causes a decrease in serum PP concentrations as a means of maintaining energy homeostasis, although more precise studies are required. The changes in serum protein profiles by green tea also demonstrate the increase in the peak intensities of hemoglobin-related multi-MS signals, suggesting the adverse side effects of green tea, although blood samples from db+/db+ mice tended to exhibit a hemolytic feature compared with those from wild-type mice (data not shown). Interestingly, the hemoglobin-related multi-MS signals shown in Fig. 9 may include hemoglobin α-chains and β-chains [9], their dimers, and possibly variously glycated hemoglobins. Hemoglobin A1c is used as a marker of diabetes in clinical diagnostic tests. Until now, however, the extent of glycation per hemoglobin that practically occurred is not clear. More detailed analyses of serum protein profiles using SELDI-TOF-MS will provide a more useful clinical index of the diabetic state. We observed that green tea improved oral glucose tolerance in humans. It is therefore likely that green tea is prophylactic against diabetes and ameliorates diabetic hyperglycemia. Green tea consumption at moderate doses may be associated with a reduced risk of type 2 diabetes in apparently healthy individuals by controlling postprandial hyperglycemia. Conclusions The control of postprandial hyperglycemia by green tea can help reduce the risk of type 2 diabetes. In the present study, we provide evidence showing that green tea promotes glucose metabolism in healthy humans, and produces an antihyperglycemic effect in diabetic mice. In addition, we analyzed the serum protein profiles of db+/db+ and +m/+m mice for the first time using SELDI-TOF-MS, and further investigated its association with any effects of oral green tea administration on serum proteins. Among the several proteins that were significantly lowered in the serum of diabetic mice, the 4211(4212) Da protein was significantly decreased after green tea administration. This is the first report demonstrating that a certain serum protein is involved in the antihyperglycemic effect of green tea. The contribution of this protein, therefore, should be further investigated in a future study. Moreover, we speculate that the observed effects of green tea on BGL are primarily due to the promotion of insulin action in peripheral tissues, such as skeletal muscles and adipocytes. Indeed, a recent paper showed that green tea supplementation for 12 weeks ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model resembling the human type 2 diabetes mellitus [3]. Since the administration of green tea produced an acute antihyperglycemic effect on BGL in diabetic mice in the present study, additional mechanisms, such as changes in amelioration or enhancement of insulin action, should be clarified in a future study. To elucidate whether new protein synthesis is required for green tea action, it would be useful to examine the influence of protein synthesis inhibitors on the acute antihyperglycemic effect of green tea. Methods Housing and care of animals C57BLKS/J db+/db+ mice (male, 9 – 11 weeks old, 36.2 – 46.3 g, BGL: 243–411 mg/dl) and its age-matched control C57BLKS/J +m/+m mice (male, 20.6–23.9 g, BGL: 110–185 mg/dl) were purchased from SLC (Shizuoka, Japan) and used as the type 2 diabetic mouse model. Male ddY mice (4 weeks old, purchased from SLC, Shizuoka, Japan) were singly injected with STZ (150 mg/kg, i.v.), and then used 4–6 weeks after the injection (28.8–38.5 g, BGL: 247–600 mg/dl). The age-matched normal ddY mice (34.7–38.6 g, BGL: 129–197 mg/dl) were also used. The animals were housed (3–5 per cage) under a daily cycle of 12 h light and 12 h darkness, with free access to food and water. Animals were treated as approved by the Toyama Medical and Pharmaceutical University Animal Research Committee, and according to the guidelines for animal experiments established by the Japanese Pharmacological Society. Sampling of blood Mice were deprived of food for 11–14 h. Blood samples (20 μl) were collected from mouse tail veins under ether anesthesia. In experiments for proteomic analyses and serum insulin measurements, blood samples (100 μl) were collected from the orbital venous plexus of mice under ether anesthesia. The food deprivation was continued throughout the measurement of blood glucose levels (until 6 h after the administration with green tea). In the oral glucose tolerance test as described below, blood samples (0.3 μl) were obtained from human skin microvessels using FreeStyle (Nipro, Osaka, Japan). Oral glucose tolerance test (OGTT) Healthy human volunteers (18–24 years old) were fasted 12 h before the starting point of experiments. The participants were perorally administered with either a suspension of green tea powder or hot water at 9:20 a.m. Ten min after the administration (at 9:30 a.m.), the participants were perorally administered with 225 ml of Trelan-G75 (Shimizu Pharmaceuticals Co., Shimizu, Shizuoka) containing 75 g glucose. BGLs were measured before and 30, 60, and 120 min after the administration with Trelan-G75 (glucose). All the subjects enrolled in this study were ethnic Japanese. Before participation, the purpose and risks of the study were carefully explained, and written informed consent was obtained from all the participants. The protocol was approved by the Toyama Medical and Pharmaceutical University Ethics Committee regulating human research. Measurement of blood glucose levels and serum insulin levels BGLs were measured using ANTSENSE II (Horiba, Kyoto, Japan) in the mouse study and using FreeStyle (Nipro, Osaka, Japan) in the human study. Serum insulin levels were measured using an insulin-ELISA kit (Morinaga Seikagaku, Tokyo, Japan). To prepare the serum samples, blood samples from mice (100 μl) were kept on ice for 2 h, centrifuged at 16,000 × g for 1 min at 4°C, and its supernatant was immediately separated from the pellet. SELDI ProteinChip Analysis Quantitative serum proteomic profiles were measured with SELDI-TOF-MS (Ciphergen Biosystems, Yokohama, Japan). The mouse serum was prepared as described above. Several types of ProteinChip Array, i.e., Q10 (an anionic exchanger, pH8), CM10 (a cationic exchanger, pH4 and pH7) and IMAC30-Cu2+ (immobilized metal affinity chromatography, pH7) (Ciphergen Biosystems), were used to fractionate proteins in serum. For the proteomic analyses with Q10 ProteinChip array, samples were 10-times diluted with denaturation buffer (7 M urea/ 2 M Thiourea/ 4% CHAPS/ 1% dithiothreitol/ 2% ampholine) and incubated on ice for 10 min, and then 10-times more diluted with buffer of 50 mM Tris-HCl (pH8). When CM10 ProteinChip array was used, samples were 10-times diluted with the denaturation buffer and incubated on ice for 10 min, and then 10-times more diluted with buffer of 100 mM NaOAc (pH4) or 50 mM HEPES (pH7). When IMAC30 ProteinChip array was used, samples were 100-times diluted with phophate-buffered saline. Spots on the different arrays were equilibrated with the buffer used for sample dilution, e.g., Tris-HCl buffer for spots on Q10 array, and each sample solution (70 μl) was loaded onto two separate spots on the arrays. After incubation for 20 min with rotation spots were rinsed with water and air-dried completely. The spots were analyzed using the SELDI ProteinChip system (PBS-IIc, Ciphergen Biosystems). In each sample, data from the two spots were averaged (duplicate assay). If a signal/noise ratio was larger than 2, the peak was considered to reflect the amount of a protein. Quantitative nature of the instrument was confirmed as previously described [14]. The m/z value of each protein peak (the molecular weight of each detected protein) was corrected, based on the m/z values of the external standards of peptides or proteins, the molecular weights of which are known. Standard errors in the estimated molecular weights were less than 0.1%. The axis of abscissa in spectra (Figs 6B, 7B, 8 and 9) indicates the mass-to-charge ratio (m/z) of protonated proteins. Reagents Fresh raw leaves of tea (Camellia sinensis, Theaceae) (Cultivar No.12, Chinhsuan and No.13, Tzuiyu) were prepared at the Taiwan Tea Experiment Station, picked in May at Nan-Tou County, and immediately used without drying. The leaves were steeped in hot water (95°C) for 30 min and the filtrate was condensed under reduced pressure. The dry weight of the extracts was determined, and the extracts were dissolved in saline for administration into mice. On the other hand, green tea powder was donated by Fukuju-en (Kyoto, Japan). The particle size (median diameter) of the green tea powder was 2.9 μm, according to the measurement using a Centrifugal Particle Size Analyzer (Shimadzu, Kyoto, Japan). In the human study, the tea powder (1.5 g) was added to 150 ml of hot water (80°C) and then whipped with a bamboo whisk. In the mouse study, the tea powder was suspended in saline at room temperature using a sonicator. The contents of caffeine and catechins in these tea samples were determined by HPLC: the samples were injected into an HPLC column (TSK-GEL, ODS-80TM, Tosoh, Tokyo, Japan); the eluent was 10 mM phosphate buffer (pH2.6)/MeCN (gradient: 5 to 15%) at a flow rate of 1.3 ml/min. The data are shown in Table 1. Table 1 Contents of catechins and caffeine in green tea samples (grams per 100 g) Green tea powder No. 12a No. 13b catechins  C 0.1 1.3 0  EC 0.6 1.9 1.0  ECg 0.6 1.6 0.2  EGC 0.3 0.9 5.8  EGCg 5.6 10.6 3.5  total 7.2 16.3 10.5 caffeine 4.3 4.5 1.7 C: (+)-catechin, EC: (-)-epicatechin, ECg: (-)-epicatechin gallate, EGC: (-)-epigallocatechin, EGCg: (-)-epigallocatechin gallate, total: total catechins. a,b The dried extract of fresh raw leaves of tea (Camellia sinensis, Theaceae) prepared at Cultivar No.12 (a, Chinhsuan) and No. 13 (b, Tzuiyu). Statistical analysis The significance of differences between two groups was assessed by Student's t-test, and the differences between multiple groups were assessed by one-way analysis of variance (ANOVA) followed by the Scheffé's multiple range test. Values of P less than 0.05 were considered to be significant. Especially, to determine the significance of the time-dependent effects of green tea (Fig. 1 and 2) on the blood glucose levels, repeated measures ANOVA was performed, and then, if the statistical significance was detected by this analysis, further statistical comparison at each measurement time between the groups was conducted by one-way ANOVA, followed by the Scheffé test. Authors' Contributions IK had the original idea for the study, designed and coordinated the experiments, and wrote the manuscript. JW prepared fresh young leaves of green tea and the hot water extract. MT assisted with measurement of blood glucose and insulin level. MI, HT and TS were involved in study design, data analysis, data interpretation and writing of the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Differences in serum protein profiles between diabetic and wild-type mice Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (left 2 panels, representative of 4–8 independent observations) and the peak intensity averages at m/z indicated (right panels; +m/+m: open column, n = 4; db+/db+: closed column, n = 8). The analyzed peak is indicated by arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak in wild-type mice, by unpaired t-test. Types of ProteinChip used were described in the Fig. 6 legend. Click here for file Additional File 2 Changes in serum protein profiles of db+/db+ mice after green tea administration MS spectra shows typical changes in the serum protein profiles of db+/db+ mice administered with saline (left) or green tea (right). Graph shows the peak intensity averages at m/z indicated, before (open column) and after (closed column) administration with saline (n = 4) or green tea (n = 4). The analyzed peak is indicated by arrows in the MS spectra. **P < 0.01; significantly different from the peak obtained before the administration, by unpaired t-test. Types of ProteinChip used were described in the Fig. 7 legend. Click here for file Acknowledgements The present work was supported in part by Fugaku's Grant by Charitable Trust (Japan). Powdered green tea was kindly donated by Fukujuen (Sourakugun, Kyoto, Japan). The donator had no role in study design, data analysis, data interpretation or writing of the manuscript. We thank Dr. Y Sugihara, Y Fukuda, and S Hara for technical assistance; Dr. R Taniguchi (CHA Research Center, Fukujuen) for measurement of particle size of green tea powder and the amount of caffeine and catechins in the tea samples; and Dr. H Tanaka and R Wakatabe (Ciphergen Biosystems KK, Yokohama Laboratory, Japan) for measurement and analysis of SELDI-TOF-MS data. ==== Refs Benelli R Vene R Bisacchi D Garbisa S Albini A Anti-invasive effects of green tea polyphenol epigallocatechin-3-galleate (EGCG), a natural inhibitor of metallo and serine proteases Biol Chem 2002 383 101 105 11928805 Weisburger JH Chung FL Mechanisms of chronic diseases causation by nutritional factors and tobacco products and their prevention by tea polyphenols Food Chem Toxicol 2002 40 1145 1154 12067577 10.1016/S0278-6915(02)00044-3 Wu LY Juan CC Hwang LS Hsu YP Ho PH Ho LT Green tea supplementation ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model Eur J Nutr 2004 43 116 124 15083319 10.1007/s00394-004-0450-x Kasahara T Kato T A new redox-cofactor vitamin for mammals Nature 1993 422 832 12712191 10.1038/422832a Wu L-Y Juan C-C Ho L-T Hsu Y-P Hwang L-S Effect of green tea supplementation on insulin sensitivity in Sprague-Dawley rats J Agric Food Chem 2004 52 643 648 14759162 Kobayashi Y Suzuki M Satsu H Arai S Hara Y Suzuki K Miyamaoto Y Shimizu M Green tea polyphenols inhibit the sodium-dependent glucose transporter of intestinal epithelial cells by a competitive mechanism J Agric Food Chem 2000 48 5618 5623 11087528 10.1021/jf0006832 Sabu MC Smitha K Kuttan R Anti-diabetic activity of green tea polyphenols and their role in reducing oxidative stress in experimental diabetes J Ethnopharmacol 2002 83 109 116 12413715 10.1016/S0378-8741(02)00217-9 Keijzers GB De Galan BE Tack CJ Smits P Caffeine can decrease insulin sensitivity in humans Diabetes Care 2002 25 364 369 11815511 Nakanishi T Koyama R Ikeda T Shimizu A Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole J Chromatogr B Analyt Technical Biomed Life Sci 2002 776 89 100 10.1016/S1570-0232(02)00078-8 Kaszkin M Beck K-F Eberhardt W Pfeilschfter J Unravelling green tea's mechanisms of action: More than meets the eye Mol Pharmacol 2004 65 15 17 14722232 10.1124/mol.65.1.15 Loew D Kaszkin M Approaching the problem of bioequivalence of herbal medicinal products Phytother Res 2002 16 705 711 12458470 10.1002/ptr.1248 Stanley S Wynne K Bloom S Gastrointestinal satiety signals III. Glucagon-like peptide 1, oxyntomodulin, peptide YY, and pancreatic polypeptide Am J Physiol Gastrointest Liver Physiol 2004 286 G693 G697 15068960 10.1152/ajpgi.00536.2003 Baetens D Stefan Y Ravazzola M Malaisse-Lagae F Coleman DL Orci L Alteration of islet cell populations in spontaneously diabetic mice Diabetes 1978 27 1 7 340309 Shiwa M Nishimura Y Wakatabe R Fukawa A Arikuni H Ota H Kato Y Yamori T Rapid discovery and identification of a tissue-specific tumor biomarker from 39 human cancer cell lines using the SELDI ProteinChip platform Biochem Biophys Res Commun 2003 309 18 25 12943657 10.1016/S0006-291X(03)01520-1

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==== Front BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-151529402510.1186/1471-230X-4-15Case ReportLiver, spleen, pancreas and kidney involvement by human fascioliasis: imaging findings Zali Mohammad Reza [email protected] Tahereh [email protected] Saeed [email protected] Azita [email protected] Ali [email protected] Research Center for Gastroenterology and Liver Diseases, Shaheed Beheshti University of Medical Sciences, PO Box 19857, 7th floor, Taleghani Hospital, Tabnak Ave, Tehran, Iran2 Department of Radiology, Shariati Hospital, Tehran University of Medical Sciences, North Kargar Ave, Tehran-Iran3 Address for correspondence Unit No 14, third floor, NO 8, Hakami closed, Motahari street, Tehran, Iran2004 4 8 2004 4 15 15 13 4 2004 4 8 2004 Copyright © 2004 Zali et al; licensee BioMed Central Ltd.2004Zali et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Fasciola hepatica primarily involves the liver, however in some exceptional situations other organs have been reported to be involved. The ectopic involvement is either a result of Parasite migration or perhaps eosinophilic reaction. Case presentation Here we report a known case of multiple myeloma who was under treatment with prednisolone and melphalan. He was infected by Fasciola hepatica, which involved many organs and the lesions were mistaken with metastatic ones. Discussion Presented here is a very unusual case of the disease, likely the first case involving the pancreas, spleen, and kidney, as well as the liver. Fasciola hepaticaeosinophiliaimaging ==== Body Background Human fascioliasis, a commonplace infection caused by a leaf-shaped Trematode Fasciolahepatica affects a human host by chance [1-4]. It seems that the rate of infection is increasing in many countries worldwide [1,5]. Human fascioliasis has to be differentially diagnosed from such hepatic and biliary diseases as acute hepatitis, neoplasm, visceral toxocariasis, biliary tract diseases, hepatic amebiasis, and infection with other liver flukes like schistosomiasis [2,5]. Diagnosis of the disease is achieved by locating the ova either in feces or duodendal drainage or by [7-9]. Imaging techniques proved to be the most useful method for confirming the diagnosis and also the follow-up of fascioliasis [6,8,10]. Small, often peripheral, nonenhancing, hypo dense nodules with tortuous, linear, branching tracts in CT scans, which decrease in size after successful therapy, are highly suggestive of the disease [8-12]. Immature flukes can produce ectopic masses or abscesses in various locations and during the acute phase of the disease, other structures such as subcutaneous tissue, heart, lungs, pleura, abdominal wall, brain, cecum, epididymis, and stomach can be involved [2,8,9]. In a particularly unusual report, direct peritoneal involvement with granuloma formation has been reported [1]. Eosinophilic reactions can be exhibited in the body as pleuritis and pericarditis [7,9]. Here, we present an extremely unusual radiologic incident of fascioliasis involving multiple organs. Case presentation In Dec 2001, a 52 year old man, who was a known case of Multiple Myeloma (MM), was presented to one of our affiliated hospitals with persistent right upper quadrant and epigastric pain, and anorexia for a period of 1 month. At the time of admission, the patient had been receiving prednisolone and melphalan for his MM, which was currently in remission. His recent condition began with tongue and facial edema two weeks before appearance of the abdominal pain. Upon physical examination, mild epigastric tenderness and a palpable liver were found. Neither icterus nor any positive sign of cardiopulmonary abnormalities were noted. Additionally, the patient did not have a fever and his peripheral lymph nodes were not enlarged. Initial laboratory findings were as follows: a hemoglobin of 10.6 g/dL, white blood cell count of 10,800/mm3 with 18% eosinophils, and a sedimentation rate of 90 mm at the end of the first hour. The total bilirubin was 0.5 mg/dL (0.2–0.8 mg/dl), alanine aminotranferase (ALT) 48 IU/L (0–40 IU/L), aspartate aminotransferase (AST) 46 IU/L (0–40 IU/L), and alkaline phosphatase (Alk P) 651 IU/L (60–140 IU/L). The eosinophilia fluctuated between 12 to 55 percent in various tests performed during the time period in question, with no unique patterns noted. Serum electrophoresis showed a monoclonal spike in the gamma region. Specific enzyme-linked immunosorbant assay (ELISA) produced a positive result for Fasciola hepatica, while the test was negative for Toxocara canis. Serologic tests for the presence of hepatitis A, B, and C viruses were negative. Blood and urine cultures were found to be sterile. Other laboratory studies, including repeated stool examinations for ova and parasites, showed no abnormalities. Chest x-rays did not demonstrate any parenchymal or pleural abnormality. Abdominal ultrasonography showed a mild hepatomegaly with multiple hypoechoic lesions in the liver. A CT scan revealed multiple but poorly defined, hypodense lesions in the liver, and a completely enlarged pancreas with mild bilateral pleural reaction, suggesting metastatic cancer (Fig. 1). In the search for a potential malignancy, diagnostic laprascopy was performed, which revealed the presence of white-colored lesions ranging from 1 to 3 cm in diameter on the surface of both lobes of the liver with mild ascites. Multiple liver and peritoneal biopsy specimens revealed fibrinoid necrosis, associated with granulomatous reaction and a high concentration of eosinophils in the liver, (Fig. 2) accompanied by markedly inflamed peritoneal tissue with eosniphilic infiltration. No malignant cells were identified and no evidence of extramedulary plasmocytoma was found. Specific staining for fungal organisms and acid-fast bacilli were negative. The ascitic fluid also had a high level of eosinophils. Endoscopic retrograde cholangiopancreatography (ERCP) failed to show any filling defect within the biliary tree. Furthermore, the patient underwent bone marrow aspiration that only indicated high eosniophilic infiltration. The patient was placed on albendazole (400 mg twice daily for 1 week). The treatment was well tolerated and the abdominal pain was improved rapidly. At the time of discharge, the patient was in good clinical condition. During a follow-up visit two months later, a second CT unexpectedly showed not only an increase in the number and size of the hypodense lesions in the liver, but also the extension of lesions into the pancreas, the spleen and both kidneys (Fig. 3). No evidence of peripheral enhancement of the hepatic lesions or ascites was documented. The patient was still experiencing upper quadrant pain on the right side. Laboratory investigations produced a white blood cell count of 7200/mm3 with 16% eosinophilia. Repeated stool examinations failed to identify ova and parasites. The patient was given triclabendazole (10 mg/kg, bid for two days). As recommended, the patient had another follow-up CT scan three months later. At that time all of his symptoms were resolved. Follow-up CT scans revealed a considerable improvement in the number and size of the lesions. At the time of the CT scan, all of his symptoms were resolved (Fig. 4A, B). At this time the WBC was 6000/mm3 with 6% eosinophils. After 5 months and in the last CT, the lesions had almost disappeared completely (Fig. 4C, D). Discussion While fascioliasis is a well-known human parasite, it sometimes produces unusual characteristics that may influence a clinician to misdiagnose the condition. In the vast majority of cases, the diagnosis is difficult in both acute and chronic phases and some important conditions such as liver abscesses and metastasis cannot be easily differentiated from fascioliasis [1,13]. Interestingly, the larvae is able to migrate to a number of ectopic locations such as subcutaneous areas, intestines, pleura, lungs, abdominal wall, brain, cecum, epididymis, stomach, pericardial, or cerebral sites, producing very unique clinical manifestations [8,9]. Serologic testing, when performed by an enzyme linked immunoabsorbent assay (ELISA) for the detection of antibodies specific to the parasite is nearly one hundred percent sensitive and specific. Thus ELISA may be used to confirm the diagnosis in acute and chronic phases [7-9]. Among imaging tools, ultrasonography is of little diagnostic value during the acute phase, while a contrast-enhanced CT scan can be very useful for diagnosis [9,17]. In CT images one can find two distinct kinds of lesions: single or multiple hypodense nodular areas caused by deposition of the parasite (abscess-like lesions) and tunnel-like branching or tortuous hypodensity which is created as the result of parasite migration through the liver and is highly suggestive of the disease [9,17]. If peripheral tortuous lesions are present, hepatic fascioliasis should be the primary diagnostic consideration [14]. As clinical and laboratory findings of fascioliasis may easily be confused with many other conditions, a high index of suspicion is required to establish a correct diagnosis [9,17]. Both CT scan and ultrasonography can be helpful in evaluating the response to treatment [15]. The ingested metacercariae of Fasciola hepatica penetrate the intestinal wall and migrate through the peritoneal cavity to reach the liver. However, ectopic migration to other locations is one of the strangest manifestations of the infection [8,9]. The precise route of migration toward ectopic sites is unclear but most often occurs in the acute stage of the disease [9]. In addition, a syndrome of eosinophilic reaction without direct parasitic involvement may accompany acute fascioliasis [9]. Ascites is not a common finding in fascioliasis, but it is not unheard of, as the peritoneum is the usual route of migration of the parasite towards the liver. Kabaalioglu A. et al reported mild splenomegaly and enlargement of the left rectus abdominalis muscle in hepatic fascioliasis [17]. In a study the authors found that among pleuro-pulmonary diseases, parenchymal infiltrates resembling the Loeffler syndrome and pleural effusion were the most common radiological features [8]. Radiologic manifestations of fascioliasis in a case with peritoneal involvement have been described as low-density lesions in the mesentery [18]. While the radiologic presentations of the disease are contradictory, reporting any new set of images related to the unusual organ involvement is of paramount importance. The case presented here is probably the first report of fascioliasis with spleen, pancreas, and kidney involvement. Migration of Fasciola hepatica to the spleen and pancreas and perhaps the kidneys was considered as the cause of the imaging findings in this patient, however hypersensitivity reaction to Fasciola antigens might also be implicated. Pathology examination to confirm the ectopic locations of the involvement was not performed, however improvement of the lesions on follow-up imaging studies after treatment defended the involvement of those regions by the parasite The compromised immunity of the patient linked to the use of immunosuppressive medicines could partly explain the extensive extra-hepatic involvement of the disease. The patient responded fully to Triclabendazole and is now healthy. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgement With special thanks to Kathleen Hekmatdoost, APRN, MSN, NP, for editing the article. ==== Refs Jimenez Bustamante J Loja Oropeza D Ruiz Semba E Maco FV Marcos RL Aviles Gonzaga R Hepatic fascioliasis: a diagnosis problem? [Abstract] Rev Gastroenterol Peru 2001 21 148 152 12172568 Dick MacLean J Graeme-Cook FM A 50-year-old man with eosinophilia and fluctuating hepatic lesions NEJM 2002 346 1232 1239 11961153 10.1056/NEJMcpc020012 Noyer CM Coyle CM Werner C Dupouy-Camet J Tanowitz HB Wiitner M Hypereosinophilia and liver mass in an immigrant Am J Med Hyg 2002 66 774 776 Teichmann D Grobusch MP Gobels K Muller HP Koehler W Suttorp N Acute fascioliasis with multiple liver abscesses Scand J Infect Dis 2000 32 558 560 11055665 10.1080/003655400458884 Haseeb AN el-Shazly AM Arafa MA Morsy AT A review on fascioliasis in Egypt J Egypt Soc Parasitol 2002 32 317 354 12049266 Cosme A Ojeda E Cilla G Torrado J Alzate L Beristain X Orive V Arenas J Fasciola hepatica. study of a series of 37 patients Gastroenterol Hepatol 2001 24 375 380 11674955 Neff GW Dinavahi RV Chase V Reddy KR Laparoscopic appearance of fasciola hepatica infection Gastrointest Endosc 2001 53 668 671 11323603 10.1067/mge.2001.113275 Pulpeiro JR Armesto V Varela J Corredoira J Fascioliasis: findings in 15 patients Br J Radiol 1991 64 798 801 1913042 Arjona R Riancho JA Aguado JM Salesa R Gonzalez-Macias J Fascioliasis in developed countries: a review of classic and aberrant forms of the disease Medicine (Baltimore) 1995 74 13 23 7837967 10.1097/00005792-199501000-00002 Andresen B Blum J Von Weymarn A Burge M Steinbrich W Duewell S Hepatic fascioliasis: report of two cases Eur Radiol 2000 10 1713 1715 11097394 10.1007/s003300000477 Han JK Choi BI Cho JM Chung KB Han MC Kim CW Radiological findings of human fascioliasis Abdom Imaging 1993 18 261 264 8508088 Sherlock S Dooley J Disease of the liver and biliary system Oxford: Blackwell Science 2002 519 Kim KA Lim HK Kim SH Lee WJ Lim JH Necrotic granuloma of the liver by human fascioliasis: imaging findings Abdom Imaging 1999 24 462 464 10475928 10.1007/s002619900540 Van Beers B Pringot J Geubel A Trigaux JP Bigaignon G Dooms G Hepatobiliary fascioliasis: noninvasive imaging findings Radiology 1990 174 809 810 2406786 Jimenez C Jimenez FJ Hoyos ML Monllau A Hepatic fascioliasis: the usefulness of CT and US Rev Esp Enferm Dig 1995 87 397 398 7626300 Carnevale S Rodríguez MI Santillán G Labbé JH Cabrera MG Bellegarde EJ Velásquez JN Trgovcic JE Guarnera EA Immunodiagnosis of Human Fascioliasis by an Enzyme-Linked Immunosorbent Assay (ELISA) and a Micro-ELISA Clinical and Diagnostic Laboratory Immunology 2001 8 174 177 11139214 10.1128/CDLI.8.1.174-177.2001 Kabaalioglu A Cubuk M Senol U Cevikol C Karaali K Apaydin A Sindel T Luleci E Fascioliasis: US, CT, and MRI findings with new observations Abdom Imaging 2000 25 400 404 10926194 10.1007/s002610000017 Raj V Nunes D Image of the month Fascioliasis Gastroenterology 1998 114 872

15294025

PMC517498

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2021-01-04 16:29:54

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BMC Gastroenterol. 2004 Aug 4; 4:15

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BMC Gastroenterol

10.1186/1471-230X-4-15

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==== Front BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central London 1471-230X-4-191534167010.1186/1471-230X-4-19Research ArticleAntroduodenal motility in neurologically handicapped children with feeding intolerance Werlin Steven L [email protected] Department of Pediatrics, The Medical College of Wisconsin and The Children's Hospital of Wisconsin Milwaukee, WI United States of America2004 1 9 2004 4 19 19 2 5 2004 1 9 2004 Copyright © 2004 Werlin; licensee BioMed Central Ltd.2004Werlin; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Dysphagia and feeding intolerance are common in neurologically handicapped children. The aim is to determine the etiologies of feeding intolerance in neurologically handicapped children who are intolerant of tube feedings. Methods Eighteen neurologically handicapped children, followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin who were intolerant of gastrostomy feedings. The charts of these 18 patients were reviewed. Past medical history, diagnoses, history of fundoplication and results of various tests of gastrointestinal function including barium contrast radiography, endoscopy and antroduodenal manometry were documented. Results Five of 11 children had abnormal barium upper gastrointestinal series. Seven of 14 had abnormal liquid phase gastric emptying tests. Two of 16 had esophagitis on endoscopy. All 18 children had abnormal antroduodenal motility. Conclusions In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. ==== Body Backround Oral pharyngeal dysphagia due to disordered swallowing has become increasingly recognized in children with cerebral palsy and other neurodevelopmental disorders. This has led to the increasing use of enteral tube feedings either for full or supplemental nutritional support. Symptoms of foregut dysmotility, such as vomiting, retching gagging and bloating, are often associated with tube feeding in neurologically handicapped children [1-4]. Previous studies have demonstrated that recurrent vomiting, aspiration and/or failure to thrive may be present in as many as 10–15% of institutionalized patients with psychomotor retardation. Antroduodenal motor function has been little studied in such children [5,6]. In order to elucidate the mechanisms behind these symptoms we reviewed the charts of a group of children followed in the Tube Feeding Clinic at the Children's Hospital of Wisconsin with neurological dysfunction, who were intolerant of tube feedings and who had undergone antroduodenal motility studies as part of their evaluations. Methods The charts of 18 neurologically handicapped children (mean age 4 years, range 1–10 years; 10 males) with dysphagia and symptoms of foregut motility were reviewed. The symptoms, underlying disorders, feeding route, the presence of fundoplication, and the use of prokinetic agents and H2 b1receptor antagonists are summarized in Table 1. All except one patient were completely or partially fed enterally. One patient ate orally but required frequent venting of his gastrostomy. Table 1 Patient population Patient Number Age (y) Diagnosis Symptoms Feeding Route UGI Gastric Emptying EGD PEG Fundoplication Other Medications H2RA Cisapride 1 9 CP GER vomiting retching oral paraesophageal hernia normal normal + gastric bezoar 2 4 feeding aversion vomiting retching gastrostomy esophageal dysmotility ND esophagitis + + + 3 3 Down's syndrome gagging retching gastrostomy normal delayed normal + 4 2 cerebral dysgenesis seizures Irritability jejunostomy GER delayed esophagitis + omeprazole 5 5 chromosome 19 deletion, GER subglotic stenosis retching gastrostomy normal normal ND + 6 4 hydrocephalus retching gastrostomy normal normal normal + + + 7 1 CP irritability gastrostomy paraesophageal hernia normal normal + + + 8 2 1/2 cerebral atrophy recurrent aspiration pneumonia vomiting gastrostomy GER normal normal + + + 9 2 diphragmatic hernia GER vomiting jejunostomy GER delayed ND + surgical jejunostomy + + 10 5 CP spina bifida retching bloating constipation jejunostomy normal normal normal + surgical jejunostomy + + 11 9 Floating Harbour syndrome retained food vomiting oral ND rapid retained food + + + + 12 17 mitochondrial disease vomiting diarrhea retching gagging bloating gastrostomy esophageal dysmotility delayed normal + + + 13 3 CP GER retching gagging gastrostomy ND normal normal + + 14 6 charge syndrome chromosome 13 Deletion retching gagging gastrostomy normal normal esophagitis + + + 15 2 CP hepatoblasoma vomiting gastrostomy paraesophageal hernia delayed normal + Nissen breakdown liver resection pyloroplasty + + 16 1 CP vomiting gastrostomy GER delayed esophagitis surgical gastrostomy - pyloroplasty + + 17 2 feeding aversion vomiting gastrostomy normal + normal + + + 18 10 CP seizures hydrocephalus vomiting retching gagging bloating TPN + + + + + GER: gastroesophageal reflux CP: cerebral palsy: NE: not done TPN: total parenteral nutiriton Following an overnight fast, antroduodenal motility studies were performed using a multilumen catheter with 8 recording ports spaced 2.5–5 cm apart, passed through the gastrostomy either under fluoroscopic guidance or endoscopically, connected to a low compliance, pneumohydraulic capillary infusion system (Arndorfer Medical Specialties, Greendale, WI) and a computerized motility system (Redtech, Calabasas, CA). Fasting activity was recorded for 3–4 hours. Erythromycin (1 mg/kg) was given intravenously over 10 minutes and the recording continued for another hour. Octreotide (0.5 mcg/kg) was then given intravenously over 5 minutes. 45 minutes later a liquid meal was given followed by an additional 2–3 hour recording period. The meal varied and consisted of the usual formula and volume given at home. Patients receiving jejunal feedings or TPN were given a bolus gastrostomy feeding. In the one patient receiving TPN, the TPN was discontinued during the study. Prokinetics were stopped at least 48 hours prior to study. Phase 1 of the MMC was defined as motor quiescence. Phase 2 was defined as the time between Phases 1 and 3 and is characterized by random contractions of varied amplitude and frequency. Phase 3 of the MMC is characterized by an aborally propagating cluster of repetitive contractions with a frequency of 11–13/minute in the duodenum and 3/minute in the antrum with a duration of 3–10 minutes. The tracings were analyzed by visual inspection. This study was approved by the Research and Publications Committee/Human Rights Review Board of the Children's Hospital of Wisconsin and the Institutional Review Board of the Medical College of Wisconsin. Results Eleven children had had recent upper GI series. Of these 5 were normal, 3 had gastroesophageal reflux and 3 had paraesophageal hernias (all following fundoplications), 1 had a bezoar and 1 had esophageal dysmotility. Fourteen patients had liquid phase gastric emptying studies. Of these 7 were normal, 6 had delayed emptying and 1 had rapid emptying. Two of 16 patients who had had recent endoscopies had esophagitis, 14 were normal. The diagnoses and clinical histories of the patients are summarized in Table 1. Twelve of the 18 patients had had a fundoplication and 9 of the 12 had had a pyloroplasty. Indications for fundoplication were frequently poorly described in the medical records but typically included vomiting and feeding intolerance. The incidence of symptoms such as retching and sweating could not be determined No patients had a normal antroduodenal motility study (Figures 1,2,3). In the fasting state 12/18 failed to have phase 3 of the MMC. Eight had predominantly phase 2 activity and did not demonstrate normal fasting phase 1. Nine had non-propagating clusters. One had MMCs that propagated in a retrograde fashion. Figure 1 An 8-year-old boy who was TPN dependent demonstrates reverse peristalsis. Note lack of antral contractions. Channel 1–2 antrum; 3–6 duodenum. Figure 2 Lack of fasting phase 3 activity during a 3 hour monitoring period in an 8-year-old boy. He also had no phase 3 like activity following erythromycin. Channel 1 antrum; 2–6 duodenum. Figure 3 Lack of responsiveness to erythromycin in a 3-year-old boy with feeding aversion. This patient also had postprandial hypomotility. Channels 1 stomach; 2–3 antrum; 4–7 duodenum. Following erythromycin eight had a normal response consisting of antral contractions with a frequency of 3/min followed by phase 3 like activity in the duodenum. Three patients had no response and seen had abnormal responses consisting of abnormal clusters in three, no antral response in three and no duodenal response in three. Fourteen patients had a normal response to octreotide consisting of cessation of antral activity and the development of phase 3 activity in the duodenum. Two patients had continued antral contractions and two did not develop phase 3 duodenal activity. These patients had non-propagating duodenal clusters. Fourteen patients had normal postprandial phase 2-like activity. Eight developed premature phase 3 activity within 30 minutes following the meal. Seven patients failed to develop phase 3 activity during the 2–3 hour postprandial monitoring period and seven had no antral contractions in the postprandial monitoring period. One patient had a retrograde MMC. Three patients developed severe pain or irritability associated with antral or duodenal contractions following erythromycin [2] or octreotide [1]. There was no correlation between any constellation of symptoms and manometric abnormalities. Conclusions Foregut dysmotility is common in children with neurodevelopmental disorders such as cerebral palsy [1-4]. Up to 75% of institutionalized children with psychomotor retardation have GER [7-11]. A number of investigators have reported that neurologically handicapped children have abnormalities of lower esophageal function [12,13]. Delayed gastric emptying is common in such patients [1,2]. Many of these patients undergo fundoplication. Continued symptoms of GER following fundoplication or the development of new symptoms such as retching and gagging suggests that a more generalized foregut motility disorder is present in many of these patients [5]. The rates of complications of surgical treatment of GER that might relate to foregut dysmotility include breakdown of the wrap (0.9–13%) and the gas bloat syndrome (1.9–8%) [14]. Other complications not reported in enough detail to estimate complication rates include dumping, and gastroparesis. Ravelli and Milla have shown that gastric electrical activity as measured by the electrogastrogram (EGG) was abnormal in 31/50 neurologically handicapped children [2]. Eleven of 18 patients who were symptomatic after fundoplication had gastric dysrhythmias. Richards et al showed that neurologically impaired children with pallor, sweating, retching or forceful vomiting preoperatively were at high risk for postoperative retching and vomiting. They hypothesized that these symptoms were indicative of activation of the emetic reflex and that children with these symptoms had a more generalized disorder than those children without such symptoms [15]. In a previous study we compared gastric electrical activity as measured by EGG in a group of neurologically handicapped children who were tolerant of their tube feedings to a group that were intolerant or symptomatic during tube feedings [16]. The percentage of children in each group who had undergone fundoplication was the same. We found that although the percentage of time that normogastria, bradygastria and tachygastria were present was not different in the 2 groups, there was a significant difference in the postprandial power between the groups. This finding suggests that symptoms present in these patients such as vomiting, retching and gagging might be due to an underlying foregut motor disorder. There have been few reports of antroduodenal motility, which have focused on neurologically handicapped children with feeding intolerance. DiLorenzo and colleagues reported that 25/28 children who remained symptomatic following fundoplication had abnormal antroduodenal motility [5]. Similar to our patients a wide variety of abnormalities were found. These authors did not report how many of their patients that had neurological handicaps. Miki et al found that fasting antroduodenal motility was abnormal in 11 neurologically impaired children with symptoms of gastroesophageal reflux [6]. Due to the nature of our patients there are some limitations in the design and interpretation of our study. Because many of our patients had had multiple formula changes, we decided to use the formula, which the child was receiving at the time of the study, so that any symptoms occurring during the studies could not be attributed to a formula change. Bolus feeds were given to all patients during the study even those who had been receiving drip feeds. The fasting state could not be recorded for 4 hours in all patients, thus it is possible that absence of phase 3 of the MMC in these patients might not be abnormal. Since normal manometry data have not been published for children, non propagating clusters may or may not be normal in our patients. Since only 12 of our 18 patients had undergone fundoplication, we agree with DiLorenzo et al [5] that these motor abnormalities were not caused by surgery, rather we believe that the underlying motility disorder was more generalized than had been recognized at the time of fundoplication. In this study we have confirmed that the incidence of foregut dysmotility is very high in neurologically handicapped children with feeding intolerance. Prokinetics and acid suppression did not resolve the symptoms in our patients. Twelve of our 18 patients had had fundoplications and two had undergone two fundoplications in unsuccessful attempts to control what had been thought to be reflux symptoms. While there is no way to know how much abnormal antroduodenal motility contributed to our patient's feeding disorders, following antroduodenal manometry a number of our patients were treated successfully with jejunal feeding, suggesting that while they had foregut dysmotility, midgut motility is normal. In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Thus in this patient population generalized foregut dysmotility may mimic reflux and the decision to perform a fundoplication should be made very cautiously and only after a complete evaluation of foregut motility particularly in children with gagging retching and forceful vomiting. Competing Interests None declared. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Ravelli AM Diagnostic and therapeutic approach to vomiting and gastroparesis in children with neurological and neuromuscular handicap J Pediatr Gastroenterol Nutr 1997 25 S34 S36 9285864 Ravelli AM Milla PJ Vomiting and gastroesophageal motor activity in children with disorders of the central nervous system J Pediatr Gastroenterol Nutr 1998 26 56 63 9443121 10.1097/00005176-199801000-00010 Sullivan PB Gastrointestinal problems in the neurologically impaired child Baillieres Clin Gastroenterol 1997 11 529 546 9448914 Giudice ED Staiano A Capano G Romano A Florimonte L Miele E Ciarla C Campanozzi A Crisanti AF Gastrointestinal manifestations in children with cerebral palsy Brain Dev 1999 21 307 311 10413017 10.1016/S0387-7604(99)00025-X DiLorenzo C Flores A Hyman P Intestinal motility in symptomatic children with fundoplication J Pediatr Gastroenterol Nutr 1991 12 169 173 2051267 Miki K Harada T Kozaiwa K Tajiri H Nagai T Kawahara H Okada A Okada S Antroduodenal motor function and gastro-oesophageal reflux in neurologically impaired children, adolescents and young adults Eur J Pediatr 1998 157 695 9727859 10.1007/s004310050913 Halpern LM Jolley SG Johnson DG A significant association with central nervous system disease in children J Pediatr Surg 1991 26 171 173 2023076 10.1016/0022-3468(91)90901-5 Reyes AL Cash AJ Green SH Booth IW Gastroesophageal reflux in children with cerebral palsy Child Care Health Dev 1993 19 109 118 8462129 Roberts KB Gastroesophageal reflux in infants and children who have neurodevelopmental disabilities Pediatr Rev 1996 17 211 212 8857200 Bohmer CJM Niezen-de Boer MC Klinkenberg-Knol EC Deville' WLJM Nadorp JHSM Meuwissen SGM The prevalence of gastroesophageal reflux disease in institutionalized intellectually disabled individuals Am J Gastroenterol 1999 94 804 810 10086670 10.1016/S0002-9270(98)00735-7 Bohmer CJM Niezen-de Boer MC Klinkenberg-Knol EC Nadorp JHSM Meuwissen SGM Gastro-oesophageal reflux disease in institutionalized intellectually disabled individuals Neth J Med 1997 51 134 139 9446923 10.1016/S0300-2977(97)00055-7 Staiano A Cucchiara S Del Giudice E Andreotti MR Minella R Disorders of oesophageal motility in children with psychomotor retardation and gastro-oesophageal reflux Eur J Pediatr 1991 150 638 641 1915516 Gustafsson PM Tibbling L Gastro-oesophageal reflux and oesophageal dysfunction in children and adolescents with brain damage Acta Paediatr 1994 83 1081 1085 7841709 Oldham KT Massey M, Stringer M, Moriquand P, Howard T Antireflux surgery, in: Pediatric surgery and urology: long term outcomes London, WB Saunders 189 211 Heikenen JB Werlin SL Brown CW Electrogastrography in gastrostomy-tube-fed Children Dig Dis Sci 1999 44 1293 1297 10489908 10.1023/A:1026666811047 Richards CA Milla PJ Andrews PLR Spitz L Retching and vomiting in neurologically impaired children after fundoplication: predictive properative factors J Pediatr Surg 2001 36 1401 1404 11528615 10.1053/jpsu.2001.26384

15341670

PMC517499

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2021-01-04 16:29:54

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BMC Gastroenterol. 2004 Sep 1; 4:19

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BMC Gastroenterol

10.1186/1471-230X-4-19

oa_comm

==== Front BMC NephrolBMC Nephrology1471-2369BioMed Central London 1471-2369-5-101534166910.1186/1471-2369-5-10Research ArticleRace/ethnicity and disease severity in IgA nephropathy Hall Yoshio N [email protected] Eloisa F [email protected] Glenn M [email protected] Jean L [email protected] Division of Nephrology, Department of Medicine, University of California San Francisco, San Francisco, CA, USA2 Department of Pathology, University of California San Francisco, San Francisco, CA, USA2004 2 9 2004 5 10 10 13 4 2004 2 9 2004 Copyright © 2004 Hall et al; licensee BioMed Central Ltd.2004Hall et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Relatively few U.S.-based studies in chronic kidney disease have focused on Asian/Pacific Islanders. Clinical reports suggest that Asian/Pacific Islanders are more likely to be affected by IgA nephropathy (IgAN), and that the severity of disease is increased in these populations. Methods To explore whether these observations are borne out in a multi-ethnic, tertiary care renal pathology practice, we examined clinical and pathologic data on 298 patients with primary glomerular lesions (IgAN, focal segmental glomerulosclerosis, membranous nephropathy and minimal change disease) at the University of California San Francisco Medical Center from November 1994 through May 2001. Pathologic assessment of native kidney biopsies with IgAN was conducted using Haas' classification system. Results Among individuals with IgAN (N = 149), 89 (60%) were male, 57 (38%) white, 53 (36%) Asian/Pacific Islander, 29 (19%) Hispanic, 4 (3%) African American and 6 (4%) were of other or unknown ethnicity. The mean age was 37 ± 14 years and median serum creatinine 1.7 mg/dL. Sixty-six patients (44%) exhibited nephrotic range proteinuria at the time of kidney biopsy. The distributions of age, gender, mean serum creatinine, and presence or absence of nephrotic proteinuria and/or hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups. Of the 124 native kidney biopsies with IgAN, 10 (8%) cases were classified into Haas subclass I, 12 (10%) subclass II, 23 (18%) subclass III, 30 (25%) subclass IV, and 49 (40%) subclass V. The distribution of Haas subclass did not differ significantly by race/ethnicity. In comparison, among the random sample of patients with non-IgAN glomerular lesions (N = 149), 77 (52%) patients were male, 51 (34%) white, 42 (28%) Asian/Pacific Islander, 25 (17%) Hispanic, and 30 (20%) were African American. Conclusions With the caveats of referral and biopsy biases, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgAN. ==== Body Background IgA nephropathy (IgAN) is the most common form of glomerulonephritis (GN) worldwide [1]. Approximately 20–30% of individuals with IgAN develop end-stage renal disease (ESRD) by 10–20 years following diagnosis [2]. Despite the increasing recognition of IgAN as a significant cause of chronic kidney disease (CKD), no specific therapies for IgAN have been developed. Glucocorticoids and fish oil have been recommended for selected patients with IgAN based on controversial results from small clinical trials [3-5]. Many patients with IgAN receive no specific therapy. Clinical reports suggest that individuals of Asian/Pacific Islander heritage are more likely to be affected by IgAN than whites, African Americans, and persons of Hispanic descent. Reports from U.S. centers have generally compared results of white and African American IgAN patients, with little or no available information on U.S. patients of Asian/Pacific Islander heritage. Studies from Japan and China have reported that more individuals with ESRD in these countries had IgAN, implying that IgAN may have a more severe disease course in certain Asian populations [6,7]. To explore whether IgAN was more common and severe among Asian/Pacific Islanders in our population, we examined clinical and pathologic data on 149 patients with IgAN and a random sample of 149 patients with other primary glomerular lesions (focal segmental glomerulosclerosis, membranous nephropathy and minimal change disease) at the University of California San Francisco (UCSF) Medical Center. Methods The records of 183 percutaneous native and transplant kidney biopsies with a diagnosis of IgAN received between November 1994 and May 2001 at the Renal Pathology Laboratory at UCSF were reviewed. Baseline demographic and clinical data included age, gender, race or ethnicity, history of kidney transplant, date of biopsy, and serum creatinine concentration at the approximate time of biopsy. In addition, the presence or absence of heavy proteinuria (≥ 3.0 g/day, with or without the nephrotic syndrome) and the presence or absence of hypertension at the time of biopsy were recorded. Major ethnic groups included white, Asian/Pacific Islander, Hispanic, African American, and other/unknown. Ethnicity was determined using information from patient health insurance forms and history provided at the time of biopsy. Any case in which Henoch-Schönlein purpura, systemic lupus erythematosus or chronic liver disease were considered likely diagnoses were excluded, as were cases of IgAN superimposed on a systemic disease involving the kidney (e.g., diabetic nephropathy). Two examiners unaware of the clinical data independently reviewed the biopsies. Biopsies displaying fewer than six glomeruli by light microscopy or insufficient immunofluorescence staining, as defined below, were also excluded. Fifteen biopsies were additionally excluded due to incomplete recovery of microscopic slides from files. Biopsies from 149 patients, including 25 kidney transplant recipients, satisfied the criteria for inclusion and provided the basis for the IgAN analytic sample. Aside from IgAN, the most commonly diagnosed primary glomerular lesions at our institution over the same time period were focal segmental glomerulosclerosis (N = 314), membranous nephropathy (N = 197) and minimal change disease (N = 147). To establish baseline race/ethnicity prevalences for our region and referral population, we collected demographic data on a computer-generated random sample of individuals with non-IgAN glomerular disease (N = 149), stratified by kidney transplant. Pathologic assessment Pathologic assessment of the IgAN native kidney biopsies was performed based on Haas' IgA nephropathy classification system [8]. All cases included in the study also met the following criteria: (1) immunofluorescence studies showing at least 2+ (scale 0 to 3+) mesangial deposition of IgA, with IgA comprising the dominant immunoglobulin deposited in the glomeruli, and (2) electron microscopy (EM) studies showing the presence of mesangial deposits. Statistical analysis Demographic and clinical data are reported as mean ± standard deviation, medians with interquartile ranges, and proportions with 95% confidence limits. Inter-ethnicity comparisons were performed using the Cochrane-Mantel-Haenzsel χ2 test for categorical variables, and analysis of variance (general linear models) or Kruskal-Wallis test for continuous variables. Two-tailed P-values <0.05 were considered statistically significant. SAS version 8.2 was used for all statistical analyses (SAS Institute, Cary, NC, USA). Results Patient clinical characteristics for the IgAN group are summarized in Table 1. Eighty-nine (60%) patients were male and 60 (40%) were female. Fifty-seven (38%) patients were white, 53 (36%) Asian/Pacific Islander, 29 (19%) Hispanic, 4 (3%) African American and 6 (4%) were of other or unknown race/ethnicity. The mean age of the IgAN patients at the time of kidney biopsy was 37 ± 14 years. Among the three main ethnic groups (whites, Hispanics, and Asian/Pacific Islanders), Hispanic patients tended to be slightly younger at the time of biopsy compared with whites and Asian/Pacific Islanders. The distributions of age and gender, however, did not differ significantly among white, Hispanic, and Asian/Pacific Islander groups. Table 1 Summary characteristics: IgA nephropathy Characteristica Total (N = 149) White (N = 57) Asian/PIb (N = 53) Hispanic (N = 29) African American (N = 4) Unknown (N = 6) P-valuec Mean age (yr) 37 ± 14 38 ± 15 37 ± 15 34 ± 14 26 ± 22 36 ± 24 0.54 Mean SCr (mg/dL) 3.1 ± 3.8 3.0 ± 2.6 3.1 ± 4.5 2.9 ± 4.2 6.3 ± 6.2 2.7 ± 2.9 0.96 Male, N (%) 89 (60) 37 (65) 29 (55) 16 (55) 3 (75) 4 (67) 0.50 Proteinuria ≥ 3g/d, N (%) 66 (44) 28 (49) 20 (37) 13 (45) 3 (75) 2 (33) 0.08 Hypertension, N (%) 74 (50) 28 (49) 28 (53) 14 (48) 2 (50) 3 (33) 0.86 Transplant, N (%) 25 (17) 4 (7) 13 (24) 8 (28) 0 (0) 0 (0) 0.15 Haas subclassd, N (%) I 10 (8) 4 (8) 5 (13) 0 (0) 0 (0) 1 (17) 0.76 II 12 (10) 5 (9) 4 (10) 2 (10) 0 (0) 1 (17) III 23 (19) 10 (19) 8 (20) 2 (10) 1 (25) 2 (32) IV 30 (24) 13 (24) 8 (20) 7 (33) 1 (25) 1 (17) V 49 (40) 21 (40) 15 (37) 10 (47) 2 (50) 1 (17) aValues represent mean ± standard deviation. bAsian/Pacific Islander cP-value refers to overall NOVA, Kruskal-Wallis test, or χ2 for comparison of white, Asian/Pacific Islander and Hispanic groups. dHaas subclass assessment performed on native kidney biopsies (N = 124). The median serum creatinine (SCr) of the IgAN cohort was 1.7 mg/dL (interquartile range 1.1–3.4 mg/dL). The median SCr of the African American group was signficantly higher (5.0 mg/dL) than the other ethnic groups; however, these calculations were based on a small sample size (N = 4) due to the low prevalence (3%) of African Americans with in our IgAN cohort. Median serum creatinine concentrations were not significantly different among white, Hispanic, and Asian/Pacific Islander groups (P = 0.64). Sixty-six patients (44%) exhibited heavy (≥ 3 g/d) proteinuria, and 74 (50%) had documented hypertension (systolic blood pressure ≥ 140 or diastolic blood pressure ≥ 90 mm Hg) at the time of kidney biopsy. The fractions of patients with heavy proteinuria and hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups. Of the 124 native kidney biopsies, the majority of cases (64%) fell into Haas subclasses IV or V, which are known independent predictors of progressive disease and poor renal outcomes [2,8]. Only 22 biopsies (18%) were graded as Haas subclasses I or II, reflecting a relatively high threshold for kidney biopsy in our referral region. The distribution of Haas subclass did not differ significantly among race/ethnicity groups. Table 2 shows the demographic characteristics of the IgAN and non-IgAN groups. Among the random sample of patients (N = 149) with non-IgAN primary glomerulopathies, 67 (45%) patients had focal segmental glomerulosclerosis (FSGS), 58 (39%) membranous nephropathy, and 24 (16%) minimal change disease. Seventy-seven (52%) patients were male, 51 (34%) white, 42 (28%) Asian/Pacific Islander, 25 (17%) Hispanic, and 30 (20%) were African American. In contrast to previous reports, the distribution of gender did not differ significantly between the IgAN and non-IgAN groups (P = 0.16) [8]. Patients in the non-IgAN group were, however, significantly older (mean age 42 ± 21 years vs. 37 ± 14 years, P = 0.006) compared to patients with IgAN. In addition, the distribution of race/ethnicity differed significantly between the two groups (P < 0.001). This association of IgAN and distribution of race/ethnicity persisted even when stratified by kidney transplant (P < 0.001 for native kidney comparison, P = 0.006 for kidney transplant recipient comparison). Table 2 Patient demographics: All glomerular lesions Characteristica IgA nephropathy (N = 149) Non-IgAN glomerular lesionsb (N = 149) P-valuec Mean age (years) 37 ± 14 42 ± 21 0.006 Male, N (%) 89 (60) 77 (52) 0.16 Transplant, N (%) 25 (17) 25 (17) 1.00 Race/ethnicity, N (%)  White 57 (38) 51 (34) <0.001  African American 4 (3) 30 (20)  Asian/PId 53 (36) 42 (28)  Hispanic 29 (19) 25 (17)  Other/Unknown 6 (4) 1 (1) aValues represent mean ± standard deviation. bFocal segmental glomerulosclerosis, membranous nephropathy, and minimal change disease. cP-value refers to overall ANOVA, Kruskal-Wallis test, or χ2. dAsian/Pacific Islander Discussion In a biopsy series of 244 patients with IgAN, Haas found fewer African Americans (in a major urban setting), similar to that noted in other U.S.-based studies of IgAN [9-11]. While limited by the size of certain ethnic groups in the study population, Haas found no significant difference in renal survival associated with "white race, black race or Hispanic origin" [8]. The reason for a lower prevalence of IgAN in African Americans relative to other kidney diseases remains unclear. The frequency of IgAN in African Americans does not appear to be influenced by the higher prevalence of the IgA2 allotype among this group [12]. In our study, the fraction of biopsies in subclasses I and II (18%) was similar to that observed by Haas (23%). However, we observed a higher proportion of biopsies in subclasses IV and V (64% vs. 31%), and a lower proportion of biopsies in subclass III (19% vs. 45%) compared with Haas, possibly reflecting a temporal trend towards a higher biopsy threshold along with intergrader measurement bias. In a study reviewing the pattern of glomerulonephritis in Singapore over the past two decades, Woo and colleagues reported that IgAN was the most common primary GN occurring in Singapore (42% of all primary GNs in the first decade and 45% in the second decade) [13]. In our biopsy population during the same period that we studied, IgAN represented 12.8% of all biopsies with primary glomerular diseases and 8.4% of all biopsies (excluding transplant biopsies for non-glomerular diseases). In China, Li reported that IgAN was the leading cause of ESRD, accounting for approximately 18% of patients [6]. In a national survey of Japanese patients with ESRD, Koyama et al. reported that 28% of new dialysis patients had IgAN listed as their primary cause of ESRD. Moreover, due to the number of additional biopsies showing chronic glomerulonephritis without immunofluorescent microscopic descriptions in the survey, the authors estimated that possibly 40% of newly registered dialysis patients in Japan might have had CKD from IgAN. In contrast, only 0.8% of incident ESRD patients in the U.S. have documented or suspected IgAN [14]. Katznelson and Cecka, using data from the United Network for Organ Sharing (UNOS), have also reported a higher incidence of IgAN and chronic glomerulonephritis causing ESRD in Asian/Pacific Islander American recipients of renal allografts between 1991 and 1996 [15]. In contrast, based on smaller biopsy series, a striking variation in prevalence rates of IgAN has been reported from Europe and South America. In the UK, for example, Ballardie and colleagues noted comparatively low prevalence rates of IgAN in a predominantly white population (Manchester, England) in the early 1970's. In the subsequent 15-year period, however, these investigators reported a phenomenal rise in the observed incidence of IgAN (accounting for 31% of all glomerulopathies in 1986), which the investigators felt more likely reflected a higher frequency of detection rather than true rise in disease incidence. Similar prevalence rates have also been documented from isolated white populations in Finland and southern Italy [16,17]. In contrast, few studies have addressed the epidemiology of IgAN in Latin America. In a small Brazilian single-center cohort (N = 205) of primary glomerular diseases, Mazzarolo et al. reported relatively modest prevalence rates (10.2%) of IgAN [18]. A larger series (N = 1,263) of renal biopsies from Peru noted much lower prevalence rates of IgAN, which accounted for only 0.9% of all glomerular lesions over a 10-year period at a central reference renal pathology laboratory in Lima [19]. These differences may be partially attributed to increased screening and disparities in the indication for kidney biopsy. In Japan and South Korea, for example, school-aged children undergo annual screening for urinary abnormalities; kidney biopsy is subsequently recommended for children with evidence of proteinuria or hematuria [20]. More comprehensive yearly health exams are further performed on full-time salaried employees throughout Japan, Singapore, and Hong Kong, making detection more likely in these ethnic groups. Furthermore, a significant reporting bias may also contribute to the higher reported prevalence rates of IgAN in Asian/Pacific Islanders, e.g., in the study by Koyama et al., only 502 (7%) of the approximate 6800 patients diagnosed with IgAN had undergone a confirmatory kidney biopsy [7]. Although the etiology of IgAN remains unknown, there exists a strong suspicion for an environmental antigen trigger combined with a genetic susceptibility factor. Along these lines, several hypotheses have been proposed to account for the reportedly higher prevalence of IgAN in Asian/Pacific Islanders. With respect to potential dietary antigens, Wakai et al. found that high intake of rice and n-6 polyunsaturated fatty acids (PUFA) were associated with an increased risk of IgAN [21]. Recent reports from Japan have also suggested a potential role of H. parainfluenzae as a causative agent of IgAN in Japanese children and adults. Such claims are supported by studies showing the glomerular deposition of outer membrane H. parainfluenzae antigens and greater levels of plasma IgA1 antibody against OMHP in Japanese patients with IgAN (compared to Japanese patients with other renal diseases) [22,23]. Whether Japanese, or Asian/Pacific Islanders in general, have higher rates of H. parainfluenzae colonization and/or infection has yet to be established. The presence of either hypertension or proteinuria ≥ 3.0 g/24 hrs at the time of diagnosis significantly correlated with worsened renal survival in IgAN, even when controlling for serum creatinine at the time of kidney biopsy [2]. We found no difference in the distribution of Haas subclass, hypertension and nephrotic proteinuria among Caucasians, Asian/Pacific Islanders, and Hispanics. Despite ongoing investigative efforts, scant data are available regarding genetic markers that may predispose individuals to progressive disease from IgAN. Recent immunogenetic studies have suggested a potential role for the T-cell receptor (TCR) in the development of immune-mediated diseases. Deenitchina and colleagues found that genetic polymorphism of the TCR constant alpha chain was associated with progression of CKD in a cohort of Japanese patients with IgAN. Although promising, such polymorphisms of the TCR gene have yet to be evaluated in large, prospective studies or by genetic analysis of familial IgAN [24]. Our results contest the assertion that IgAN follows a more severe course in individuals of Asian/Pacific Islander descent. One reason for the similar disease severity of IgAN in our study population may stem in part from the large subpopulation of Filipino patients comprising our Asian/Pacific Islander cohort. It is unclear whether certain subpopulations of Asian/Pacific Islanders, including Filipinos, exhibit IgAN prevalence rates similar to those documented by Koyama and Woo. Anecdotal reports from Thailand and India documenting prevalence rates of 4–9% suggest that IgAN may not have the same epidemiology among all southeast Asians [1]. Despite having higher incidence rates of ESRD than the U.S. white population, the Asian/Pacific Islanders remain a largely unstudied group, for whom more comprehensive data collection is warranted. There are several important limitations to this report. As with any single-center biopsy series, we may have been underpowered to detect a clinically significant difference due to the limited sample size (type II error). In addition, racial admixture may have also confounded the results, as we were unable to subclassify patients in the Asian/Pacific Islander group or account for the growing population of bi- or multi-ethnic individuals in our population. Furthermore, due to the study's case control design, and breadth of our referral base (northern California and Hawaii), we were unable to control for the criteria for kidney biopsy. As a result, a biopsy bias may have confounded our results. In other words, Asian/Pacific Islander patients in our referral base with mild to moderate proteinuria and/or hematuria might have been given a presumptive diagnosis of IgAN without nephrology referral or confirmatory kidney biopsy. With regard to disease prevalence, these potential referral and biopsy biases based on race/ethnicity are largely conservative in nature, and would have biased our results towards the null. Finally, we have included data from a modest-sized IgAN transplant population (N = 25), the donor demographics of which were unavailable at the time of the study. However, the association of race/ethnicity and distribution of glomerular lesion persisted, even when stratified by kidney transplant, and thus our overall conclusions remained the same. In addition, a small European study of donor-recipient pairs (average follow-up 7 years) has shown that when a donor kidney with asymptomatic IgA deposits is transplanted into a recipient with ESRD secondary to a disease other than IgAN, the IgA immune deposits in the donor kidney are rapidly removed [25]. Conclusions In conclusion, with the caveats of referral bias and biopsy bias, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgA nephropathy. Competing interests None declared. Authors' contribution YH designed the study, collected and analyzed the data, and drafted the manuscript. GC supervised the study design, analyzed the data, and edited the manuscript. EF graded the IgAN histopathologic slides. JO collected, reviewed and graded all histopathologic data, and edited the manuscript. All authors approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments Dr. Hall is supported by the American Kidney Fund, Clinical Scientist in Nephrology Award. Dr. Chertow is supported by NIH NIDDK RO1 DK58411-01. ==== Refs Levy M Berger J Worldwide perspective of IgA nephropathy Am J Kidney Dis 1988 12 340 347 3055955 D'Amico G Natural history of idiopathic IgA nephropathy: Role of clinical and histologic prognostic factors Am J Kidney Dis 2000 36 227 237 10922300 Pozzi C Bolasco PG Fogazzi GB Andrulli S Altieri P Ponticelli C Locatelli F Corticosteroids in IgA nephropathy: a randomised controlled trial Lancet 1999 353 883 887 10093981 10.1016/S0140-6736(98)03563-6 Donadio JV Bergstralh EJ Offord KP Spencer DC Holley KE A controlled trial of fish oil in IgA nephropathy N Engl J Med 1994 331 1194 1199 7935657 10.1056/NEJM199411033311804 Dillon JJ Fish oil therapy for IgA nephropathy: efficacy and interstudy variability J Am Soc Nephrol 1997 8 1739 1744 9355077 Li L End-stage renal disease in China Kidney International 1996 49 287 301 8770982 Koyama A Igarashi M Kobayashi M Natural history and risk factors for immunoglobulin A nephropathy in Japan Am J Kidney Dis 1997 29 526 532 9100040 Haas M Histologic subclassification of IgA nephropathy: A clinicopathologic study of 244 cases Am J Kidney Dis 1997 29 829 842 9186068 Korbet SM Rosangela MG Borok RZ Schwartz MM The racial prevalence of glomerular lesions in nephrotic adults Am J Kidney Dis 1996 27 647 651 8629623 Jennette JC Wall SD Wilkman AS Low incidence of IgA nephropathy in blacks Kidney International 1985 28 944 950 4087701 Galla JH Kohaut EC Alexander R Mestecky J Racial differences in the prevalence of IgA-associated nephropathies Lancet 1984 2 522 6147580 10.1016/S0140-6736(84)92599-6 Crowleg-Nowick PA Julian BA Wyatt RJ Galla JH Wall BM Warnock DG Mestecky J Jackson S IgA nephropathy in blacks: Studies of IgA2 allotypes and clinical course Kidney International 1991 39 1218 1224 1680208 Woo KT Chiang GS Pall A Tan PH Lau YK Chin YM The changing pattern of glomerulonephritis in Singapore over the past two decades Clin Nephrol 1999 52 96 102 10480220 U.S. Renal Data System, USRDS 2002 Annual Data Report Atlas of End-Stage Renal Disease in the United States National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 2002 Katznelson S Cecka JM The great success of Asian kidney transplant recipients Transplantation 1997 64 1850 1852 9422431 10.1097/00007890-199712270-00038 Ballardie FW O'Donoghue DJ Increasing frequency of adult IgA nephropathy in the UK? Lancet 1987 2 1205 2890823 10.1016/S0140-6736(87)91336-5 Donadio JV Grande JP IgA nephropathy N Engl J Med 2002 347 738 748 12213946 10.1056/NEJMra020109 Mazzarolo Cruz HM Cruz J Silva AL JrSaldanha LB de Oliveira Penna D Prevalence of adult primary glomerular diseases: retrospective analysis of 206 kidney biopsies (1990–1993) Rev Hosp Clin Fac Med Sao Paulo 1996 51 3 6 8762646 Hurtado A Escudero E Stromquist CS Urcia J Hurtado ME Gretch D Watts D Russell K Asato C Johnson RJ Distinct patterns of glomerular disease in Lima, Peru Clin Nephrol 2000 53 325 332 11305805 Kitagawa T Lessons learned from the Japanese nephritis screening study Pediatr Nephrol 1988 2 256 263 3153020 Wakai K Nakai S Matsuo S Kawamura T Hotta N Maeda K Ohno Y Risk factors for IgA nephropathy: a case-control study with incident cases in Japan Nephron 2002 90 16 23 11744800 10.1159/000046309 Ogura Y Suzuki S Shirakawa T Masuda M Nakamura H Iijima K Yoshikawa N Haemophilus parainfluenzae antigen and antibody in children with IgA nephropathy and Henoch-Schonlein nephritis Am J Kidney Dis 2000 36 47 52 10873871 Suzuki S Nakatomi Y Sato H Tsukada H Arakawa M Haemophilus parainfluenzae antigen and antibody in kidney biopsy samples and serum of patients with IgA nephropathy Lancet 1994 343 12 16 7905040 10.1016/S0140-6736(94)90875-3 Deenitchina SS Shinozaki M Hirano T Ando T Hirakata H Kiyohara Y Katafuchi R Fujishima M Association of a T-cell receptor constant alpha chain gene polymorphism with progression of IgA nephropathy in Japanese patients Am J Kidney Dis 1999 34 279 288 10430975 Koselj M Rott T Kandus A Vizjak A Malovrh M Donor-transmitted IgA nephropathy: long-term follow-up of kidney donors and recipients Transplant Proc 1997 29 3406 3407 9414767 10.1016/S0041-1345(97)00957-3

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-551533101710.1186/1471-2407-4-55Research ArticleNo evidence for involvement of SDHD in neuroblastoma pathogenesis De Preter Katleen [email protected] Jo [email protected] Jasmien [email protected] Caroline [email protected] Jöel [email protected] Annick [email protected] Geneviève [email protected] Valérie [email protected] Roy Nadine [email protected] Frank [email protected] Coster Rudy [email protected] Marleen [email protected] Paepe Anne [email protected] Frank [email protected] Center for Medical Genetics, Ghent University Hospital, K5, De Pintelaan 185, B-9000 Ghent, Belgium2 Department of Pathological Anatomy, Ghent University Hospital, BLOK A, De Pintelaan 185, B-9000 Ghent, Belgium3 Department of Paediatrics, Ghent University Hospital, K6, De Pintelaan 185, B-9000 Ghent, Belgium4 Molecular Oncology Unit, Centre Léon Bérard, 28 rue Laennec, F-69373 Lyon, France2004 24 8 2004 4 55 55 29 4 2004 24 8 2004 Copyright © 2004 Katleen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma. Methods SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria. Results Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype. Conclusions Our study provides no indications for 2-hit involvement of SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis. ==== Body Background Neuroblastoma (NB) is the most frequent extra-cranial solid tumour in children, originating from immature neural crest cells of the sympathetic nervous system [1]. The tumours show remarkable differences in clinical presentation ranging from localized to highly metastatic. Although age and clinical stage are strong prognostic indicators, particular genetic aberrations, i.e. MYCN amplification and 17q gain, also have a profound predictive power [2,3]. Presently, three major clinico-genetic NB patient subgroups have been recognized (subgroup 1, 2A and 2B) [4]. Subgroup 1 consists of NB patients with favourable disease stage (stage 1, 2 and 4S), most often infants younger than one year of age presenting with tumours with a near triploid DNA content and a characteristic pattern of chromosomal instability including the consistent presence of an extra chromosome 17. The two other NB patient groups represent mainly older children with high-stage disease (stage 3 and 4) and poor prognosis. Both NB subgroups present with 17q-gain, but are distinguished by presence of MYCN amplification and 1p-deletion in subgroup 2B and 11q-deletion often in combination with 3p-deletion in subgroup 2A [3,5-9]. The first evidence for the occurrence of 11q-deletions in NB was obtained in 1991 [10]. However, it was not until recently that a specific patient subgroup with this particular genetic defect was recognized, representing approximately 20% of cases [5-9,11-13]. The recurrent finding of 11q-deletions in NB suggests the presence of a tumour suppressor gene residing on the long arm of chromosome 11. Additional functional evidence for this hypothesis came from the observation that differentiation of NB cells can be induced by transfer of an intact chromosome 11 into a NB cell line [14]. Although both comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies indicate that the majority of the 11q-deletions are distal losses encompassing a large portion of the long arm [5-9,12,13,15,16], detection of rare small or interstitial deletions allowed the provisional localization of an SRO (shortest region of overlap) at 11q23.3 between markers D11S1340 and D11S1299, encompassing a distance of approximately 3 Mb [16]. When a single tumour with two small interstitial deletions is not taken into consideration, the SRO is defined by a small subset of tumours and spans 18 Mb between markers D11S898 and D11S1299 (according to UCSC Genome Browser, freeze version July 2003). This region harbours SDHD, which encodes the small subunit D (cybS, cytochrome b558) of the mitochondrial respiratory chain complex II (succinate-ubiquinone oxidoreductase) [17,18] and was recently recognized as a prototype tumour suppressor gene [19]. The first evidence for a role of SDHD in tumour development was obtained by the discovery of germline mutations in this gene as the cause for familial paraganglioma (PGL) [19]. Somatic and occult germline SDHD mutations were also detected in patients with apparently sporadic pheochromocytoma (PC) [20,21]. It seems that most of the individuals with PC possess SDHD mutations in the 5' portion of the gene causing complete disassembly of complex II, whereas PGL are associated with mutations in the 3' region of the gene causing partial inactivation of its catalytic activity [19-28]. PGL and PC are histologically related to NB as they are all neural crest derived. NBs consist of immature neuroblasts, whereas PGL and PC contain mature chromaffin cells. Of further interest is the fact that, in addition to the well established role of SDHD in oxidative phosphorylation, SDHD has also been presumed to contribute to the function of the mitochondria as oxygen sensors. It was shown that SDHD inactivation leads to a pseudo-hypoxic state and upregulation of hypoxia responsive genes, possibly through increased production of reactive oxygen species (ROS) [23]. A hypoxia-induced shift toward a neural crest-like phenotype has been shown to result in more aggressive NB cells with increased potential to metastasize [29]. Consequently, inactivating SDHD mutations or reduced activity of SDHD might lead to impaired oxidative phosphorylation and hypoxia and thus contribute to NB oncogenesis. In view of the above, we considered SDHD as a positional and functional candidate for the presumed NB tumour suppressor gene on 11q23. In order to search for evidence for involvement of SDHD in NB development, an extensive series of investigations was performed on the DNA, RNA and protein level. Methods NB patient and cell line samples Neuroblastoma (NB) tumour samples (at least 70% tumour cells) were collected at the Ghent University Hospital (Ghent, Belgium) (n = 32) and in the Molecular Oncology Unit (Lyon, France) (n = 35). Ethical approval was obtained for the collection of the tumour samples. The latter group includes selected patients with stage 3 or 4 NB without MYCN amplification. For all NB patients constitutional leukocyte DNA was available. In addition, 31 NB cell lines were included in the analysis of which karyotypes were available. For 20 of these cell lines, comparative genomic hybridization (CGH) data and/or M-FISH (multicolour fluorescence in situ hybridization) results have been published [30-32]. For screening of sequence variants in a normal population, leukocyte DNA from 135 unrelated healthy individuals was used. DNA was extracted as previously described [33]. Cultures of NB cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP were treated with puromycine (100 μg/ml) during 6 hours in order to prevent possible nonsense mediated RNA decay of variant SDHD transcripts. RNA of the cell line pellets (treated and untreated) was extracted with the RNeasy Mini kit (Qiagen) according to the manufacturer, followed by RNase free DNase treatment on column (Qiagen). A fraction of the untreated NB cell line cultures was also used for functional enzyme assays. 11q23 status of samples Determination of the 11q23 status in NB cell lines and tumours with LOH or FISH The 11q status of the cell lines was evaluated using FISH. FISH was performed using the LSI MLL (11q23.3) SpectrumOrange probe (Vysis) and BAC clone RP11-93E4 for the CRTAM gene (11q24.1) in combination with a centromeric probe for chromosome 11. Labelling and FISH was performed as described [34]. For each case at least twenty metaphase chromosomes and 100 interphase nuclei were screened. All NB patients were analyzed with 4 microsatellite markers on 11q23: D11S1986 (11q23.1), D11S1998 (11q23.3), D11S1356 (11q23.3) and D11S1299 (11q23.3), of which D11S1986 and D11S1998 are immediately flanking the SDHD gene. In order to discriminate between whole chromosome loss, and unbalanced 11q loss (= partial 11q loss), two microsatellite markers on 11p (D11S922 on 11p15.5 and D11S1324 on 11p14.1) were analyzed in patients that showed allelic imbalance for all 11q markers (positions of the markers are according to the UCSC Genome Browser, freeze version July 2003). Scoring of loss of heterozygosity (LOH) was performed by calculation of the allelic imbalance factor (AIF) [35], whereby AIF > 2 denotes allelic imbalance, and AIF > 5 denotes LOH. Experimental conditions for the fluorescent based LOH screening can be obtained from the authors upon request. Homozygous deletion screening in NB cell lines Real-time quantitative PCR primers were designed in the four exons of SDHD using Primer Express v2.0 (Applied Biosystems) (Table 1). Exon 1 was too small for primer design in the exonic region; therefore primers flanking the exonic region were designed. Real-time quantitative PCR and quantification was performed as described [33]. MSP On the 31 NB cell lines and on another series of 50 NB tumours of which 15 were included in the mutation analysis, methylation-specific PCR (MSP) was performed as described, with minor modifications [36]. MSP primers were designed using the web-based MSP design software MethPrimer [37] and checked for specificity using the methBLAST software (Pattyn et al., in preparation). Primers were designed in a CpG island close to the start of the gene (putative SDHD promotor) (chr11: 111495002–111495330: UCSC Genome Browser freeze version July 2003) (methylated forward 5'GTAGTCGGGATCGAGTATTAGTGAGTC3', methylated reverse 5'AATAAACCGAAAATCGAAAAACGAT3', unmethylated forward 5'AGTTGGGATTGAGTATTAGTGAGTTGT3', unmethylated reverse 5'ACTAAATAAACCAAAAATCAAAAAACAAT3'). Amplification mixtures (50 μl) for the PCR reaction contained 50 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 6 mM MgCl2, 200 μM of each dNTP, 1.25 U Platinum Taq polymerase (Invitrogen), 3% DMSO and 300 nM of each primer. The cycling conditions comprised 4 min polymerase activation at 93°C, 40 cycles with denaturation at 93°C for 30 sec, annealing at 64°C (methylated primers) or 65°C (unmethylated primers) for 30 sec and extension at 72°C for 30 sec, and a final extension for 5 min at 72°C. SssI methylase (New England Biolabs) treated DNA, following the manufacturer's instructions and normal human genomic DNA were used as a positive and negative control respectively after bisulfite modification. Mutation analysis DHPLC analysis Intronic primers flanking the SDHD exons were designed using Primer Express v2.0 (Applied Biosystems), based on the publicly available SDHD genomic sequence (accession number AB026906) (Table 2). PCR reactions were performed on a PTC-200 DNA engine (MJ Research). Amplification mixtures (25 μl) contained 10 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 2.5 mM MgCl2, 200 μM of each dNTP, 1 U Platinum Taq polymerase (Invitrogen) and 500 nM of each primer. The cycling conditions comprised 3 min polymerase activation at 94°C, 35 cycles with denaturation at 92°C for 20 sec, annealing at 60°C for 20 sec and extension at 72°C for 2 min, a final extension for 5 min at 72°C and a slow decrease in temperature to 25°C over 30 minutes. One μl of the PCR products was analyzed on a Ready-To-Run Agarose Gel (1.2%) (Amersham Biosciences). Denaturing high-pressure liquid chromatography (DHPLC) was performed using the Wave system (Transgenomic). The melting profile of each fragment was determined using the Wavemaker software v4.1 (Transgenomic). Crude PCR product was injected into a preheated, fully equilibrated chromatographic column for the DHPLC analysis. Exon 1 fragments were eluted at a temperature of Tm(= 62.1°C)+0.7°C and Tm+1.5°C. Exon 2 fragments were eluted at a temperature of Tm(= 55.7°C)+4.8°C. Exon 3 fragments were eluted at a temperature of Tm(= 57.4°C)-0.4°C, Tm+1.1°C and Tm+3.8°C. Exon 4 fragments were eluted at a temperature of Tm(= 56.4°C)-0.4°C, Tm+1.6°C and Tm+3.2°C. Elution of the fragments was performed using standard conditions according to the manufacturer. Elution profiles were analyzed using the Wavemaker software. Sequencing Sequencing was performed on all cell lines and on tumour samples with aberrant DHPLC elution peaks (except from the noncoding region of exon 4 that was sequenced in all NB cell lines without preceding DHPLC mutation screening). Amplified fragments were purified using the Montage PCR96 filter plates (Millipore) or by excision of the fragment of interest from a 1.5% TBE-agarose gel and purification on a GenElute Minus EtBr Spin Column (Sigma-Aldrich). Cycle sequencing was performed using purified amplicons (3–10 ng), the above-mentioned primers (Table 2) at a concentration of 80 nM and the ABI PRISM BigDye Terminators v3.0 Cycle Sequencing Kit (Applied Biosystems), with the following thermocycling conditions: 25 cycles at 92°C for 10 sec, 55°C for 5 sec and 60°C for 3.5 min. The products were run on an automated sequencer ABI3100 (Applied Biosystems) after isopropanol precipitation. Sequence analysis was performed with the SeqScape v1.1 software (Applied Biosystems). Allelic discrimination screening for 2 sequence variants using MGB probes PCR primers and minor groove binder (MGB) probes for sequence variant IVS4-32T>C were designed using Primer Express v2.0 following the user bulletin guidelines for the design of MGB probes (Applied Biosystems): forward primer 5'TTTTTTGCAGCCAAGTTATCTGTATAG3', reverse primer 5'TGTCCAAGGCCCCTAAAGAA3', MGB probe allele 1 5'TGTGGTTTTTtATTGATG3' labelled with 6-FAM and MGB probe allele 2 5'TGTGGTTTTTcATTGAT3' labelled with VIC. To address the frequency of the sequence variant g.7876A>G (Y93C) in a normal population, the following primers and probe were designed: forward primer 5'GGCTGCTTATTTGAATCCTTGCT3', reverse primer 5'ACTTGCCAGTGACCATGAAGAGT3' and MGB probe variant allele 5'ATGGACTgTTCCCTG3' labelled with VIC. The reaction mixture contained 10 ng of DNA, 100 nM of each MGB probe, 300 nM of each primer and 1× qPCR Mastermix (Eurogentec). For the screening of the g.7876A>G variant, multiplex PCR was performed using primers and probe of the normal allele of the above-mentioned SNP (IVS4-32T>C) and primers and probe for the variant allele g.7876A>G. Reactions were performed on the iCycler Thermal Cycler (Bio-Rad) with the following thermocycling conditions: an initial activation step at 95°C for 10 min, 50 cycles of 95°C for 15 sec and 60°C for 1 min. Allelic discrimination data analysis was performed on the iCycler IQ Optical System Software v3.0a (Bio-Rad). The SNP info for the IVS4-32T>C variant was submitted to NCBI's SNP database (sn#5606973, SDHD_IVS4-32). Full-length SDHD mRNA amplification In order to investigate predicted or putative splice variants caused by the 4 bp-deletion in NB cell line N206 and SDHD sequence variants present in other cell lines, the coding region of the full-length SDHD mRNA was amplified for cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP, before and after puromycin treatment. RNA extraction, DNase treatment and cDNA synthesis were performed as described [38]. Subsequent PCR was performed with forward primer 5'AGGAACGAGATGGCGGTTCTC3' (exon 1) and reverse primer 5'GCTTCCACAGCATGGCAACA3' (exon 4). PCR products were run on an agarose gel, purified and sequenced using the above-mentioned protocol. The sequence information also provided evidence on the allelic mRNA expression status of SDHD. Quantification of SDHD expression using real-time quantitative RT-PCR Relative SDHD expression levels were determined using an optimized two-step SYBR Green I RT-PCR assay [38] with minor modifications in 31 cell lines, 7 normal control samples (human brain, trachea, lung, heart, breast, kidney, liver) and in laser capture microdissected foetal neuroblast cells [39]. The comparative CT method was used for quantification. PCR reagents were obtained from Eurogentec as SYBR Green I mastermixes and used according to the manufacturer's instructions. Primers in exon 3 were designed using Primer Express (see Primers Exon 3 in Table 1). Reactions were run on an ABI5700 (Applied Biosystems). Gene expression levels were normalized using the geometric mean of the 4 most stable internal control genes in NB (i.e. UBC, HPRT1, SDHA and GAPD) as reported previously [40]. Complex II activity and protein assays Enzyme activities were determined spectrophotometrically as previously described [41]. Protein amount was determined with immunoblot analysis of complex II Fp fragment as previously described [42]. Relative protein amounts of complex II compared to complex IV were measured using the TotalLab software (Amersham Biosciences). Ultrastructural analysis of mitochondria in NB cell lines Mitochondria of NB cell lines LA-N-2, SK-N-AS, CLB-GA, NGP, CHP-901, SK-N-SH, SK-N-FI, N206, SJNB-12, SJNB-8, NMB, SJNB-10 and IMR-32, breast carcinoma cell line MCF-7 and, Ewing sarcoma cell line SK-N-MC were analysed by electron microscopy. Monolayers from these cell lines were briefly rinsed twice in PBS and then immersed at room temperature in 3 % glutaraldehyde buffered with Na-cacodylate at pH 7.3 for 1 h. After rinses in this buffer with 1 % bovine serum albumin, cells were scraped off with a rubber policeman, and centrifuged with 3 % glutaraldehyde. After washing, the pellets were postfixed in 2 % buffered OsO4 for 1 h at 4°C. Block staining in uranylacetate (UAc) in 70 % ethanol was followed by dehydration in ethanol and propylene oxide, and embedding in Epon. Ultrathin sections were counterstained with UAc and lead. At least 2 cells in each culture were photographed at a magnification of 20,000 in a Zeiss electron microscope operating at 50 KV. Per culture, 25–63 mitochondria were examined. Their projected length (largest straight distance) was measured and matrix electron density was compared to the surrounding cytosol. Electron microscopic files were made on 11 archived neuroblastic tumours. In addition, previously published cases were examined for mitochondria morphology. Results SDHD deletion, mutation and methylation analysis 11q23-deletion screening Previous karyotyping, comparative genomic hybridization (CGH) and/or M-FISH revealed 11q-deletions in 9 out of 31 neuroblastoma (NB) cell lines (CLB-GA, GI-ME-N, IMR-32, LA-N-6, NBL-S, NGP, SK-N-AS, NMB, N206) [30-32]. In this study, the presence of 11q23-deletions was confirmed by FISH in all these cell lines except N206 for which the deletion was located distal to the MLL locus (11q23.23) (not shown) (Table 3). Sequencing analysis of SDHD on 11q23 demonstrated that the allelic imbalance in NMB does not cause loss of heterozygosity (see later). No previously unnoticed submicroscopic 11q23-deletions were detected. Screening for homozygous deletions in all SDHD exons was negative for the 31 NB cell lines. In 20 of the 67 NB tumour samples, loss of heterozygosity (LOH) or allelic imbalance (AI) (AIF > 2) in the 11q23 region was found (Table 3): unbalanced 11q LOH (i.e. partial allelic loss of the long arm of chromosome 11) in 2/32 patients of the Ghent University Hospital (Ghent, Belgium) and in 7/35 patients of the Molecular Oncology Unit (Lyon, France) and loss of markers on both chromosome arms (indicating whole chromosome 11 loss, or co-occurrence of 11q and 11p allelic loss) in 3/32 patients of the Ghent University Hospital and 8/35 patients of the Molecular Oncology Unit (Table 3). The higher frequency of chromosome 11 LOH in the patient subgroup of the Molecular Oncology Unit can be explained by the selection for patient samples of high stage without MYCN amplification, in contrast to the other patient subgroup for which samples were unselected. Mutation analysis Denaturing high performance liquid chromatography (DHPLC) analysis and subsequent sequencing of the SDHD gene in 31 NB cell lines and 67 NB tumour samples revealed the presence of sequence variants in 5 NB cell lines and 4 NB tumour samples (Table 4). Two variants were considered as bona fide mutations (Figure 1). The first, a Y93C missense mutation in cell line NMB, was not detected in 135 unrelated healthy individuals. The second variant detected in NB cell line N206, represented a 4 bp deletion on the exon-intron boundary causing an exon 3 skip leading to a premature stop codon. Interestingly, both effects are located within regions that are frequently affected in paraganglioma (PGL). Unfortunately no normal or primary tumour material of the patients from which the N206 and NMB cell lines were derived was available to test whether these are germline or somatic mutations. In one patient without 11q allelic loss (F11) we observed in both tumour and constitutional DNA a TCTA insertion at position IVS2+37. However, no additional tumour material nor parental material was available for further analysis. So, it remains unclear whether this is a true mutation or a rare polymorphism. In addition, 1 new and 4 known polymorphisms were observed. The H50R variant found in cell line LA-N-2 was described as a polymorphism in several studies [43-45]. This is also true for the G12S change found in tumour and constitutional DNA of patient F18 [21]. The previously reported polymorphisms IVS3-29A>G [25] and S68S [25,27,28,44,46,47] were detected in cell lines NGP, NMB and SK-N-FI, in both tumour and constitutional DNA of patients F18 and F35, and in constitutional DNA of patient F22. In all cases, these last two polymorphism (IVS3-29A>G and S68S) were present together with the IVS4-32T>C variant, previously described by Taschner and colleagues [28]. Allelic discrimination screening in 135 unrelated individuals revealed an incidence of the IVS4-32T>C polymorphism of 4.4% (= 6/135; allele frequency 2.2%). This is similar to the incidence found in NB cell lines (3/31 = 9.7%) and NB patient constitutional DNA (3/67 = 4.5%, allele frequency = 2.2%). The presence of the IVS3-29A>G, S68S and IVS4-32T>C variants in a cell line (NGP) and two tumours (F18 and F35), in which one of both SDHD alleles has been deleted, indicates that all three variants are located on the same allele, representing a low frequent haplotype. MSP analysis SDHD promotor hypermethylation was tested for 31 NB cell lines and 50 NB patients using methylation-specific PCR (MSP). No evidence for methylation was obtained in any of the analyzed NB cases. Analysis of the 4 bp deletion in the cell line N-206 Amplification of the full-length SDHD cDNA showed that a 4 bp deletion in the intron-exon boundary in cell line N206 caused skipping of exon 3 leading to a premature stop codon. No alternative transcripts could be detected in cell lines NMB, SK-N-FI, NGP and LA-N-2 carrying basepair variants (and 3 control cell lines without sequence variants SK-N-SH, SK-N-AS and CLB-GA) when grown with or without puromycin (Figure 1). The above-mentioned cDNA transcript sequencing revealed that SDHD is bi-allelically expressed, thus supporting recent observations in lymphoblastoid cell lines, adult kidney and adult and fetal brain [19,22], but in contrast with the initially reported paternal mono-allelic expression in PGL tissue [22]. SDHD mRNA expression analysis SDHD expression levels were measured using real-time quantitative PCR in 31 NB cell lines, normal foetal neuroblast cells (16, 18 and 19 weeks gestational time) and 7 normal adult tissues (brain, heart, kidney, liver, lung, trachea and breast) (Figure 2). The SDHD mRNA level was significantly lower in NB cell lines compared to both normal neuroblast cells (Mann-Whitney test: P = 5.31E-06) and normal adult tissue mRNA samples (Mann-Whitney test: P = 1.49E-05). SDHD mRNA levels were significantly reduced in cell lines with 11q allelic loss and SDHD mutated cell lines (i.e. NMB and N206) (N = 9) compared to cell lines without 11q allelic loss (N = 22) (Mann-Whitney test: P = 1.49E-03). SDHD functional analysis As the SDHD gene encodes the small subunit D of the mitochondrial respiratory chain complex II we decided to assess the effect of the basepair variants on the activity of complex II of the respiratory chain by spectrophotometrical measurements in 5 NB cell lines (N206, NMB, SK-N-FI, NGP and LA-N-2) and 3 control NB cell lines without sequence variants (SK-N-SH, SK-N-AS and CLB-GA). No significant differences in complex II enzyme activity could be demonstrated. Although, in LA-N-2 a slight decrease of complex II activity was observed (data not shown). On above-mentioned cell lines and NB cell lines CHP-901, SJNB-12, SJNB-8, SJNB-10 and IMR-32, breast cancer cell line MCF7 and Ewing sarcoma cell line SK-N-MC, immunoblotting of the Fp fragment of complex II showed no significant variation in abundance among the tumour cell lines (data not shown). Ultrastructural morphology of mitochondria in NB cell lines Electron microscopic analysis of NB cell lines revealed that the morphology of the mitochondria is heterogeneous between the different cell lines, with respect to length, dilated intracrista spaces and condensation of the matrix (Table 5 and Figure 3). In most of the cell lines the electron dense matrix granules are absent. A striking observation are dilations of the mitochondrial intracrista spaces in most of the NB cell lines including N206 (Figure 3A), but not NMB (Figure 3B). Cell line LA-N-2 shows very large mitochondria (Figure 3D). However, these observations are not the same as described for PGL, where swollen mitochondria are seen with an empty matrix and short or absent cristae [48]. In order to examine whether dilated mitochondrial cristae are a feature of many, or all NBs, we studied the mitochondria in electron micrographs from 11 archived and 22 previously published neuroblastic tumours [49-53]. Dilated cristae were seen in 11 tumours but they were limited to a minority of the mitochondria (2–23%), in contrast to several of the cell lines of which most mitochondria are altered. In several of the analyzed NB tumours, partially vacuolated matrices were occasionally observed. Discussion In this study, we investigated the possible involvement of SDHD in neuroblastoma (NB) tumourigenesis. In a first step, mutation and methylation analyses were performed on a large panel of NB cell lines and tumours. A total of seven sequence variants (in nine different samples) were detected of which two could represent bona fide mutations, i.e. missense mutation Y93C in cell line NMB and a 4 bp deletion in cell line N206. The Y93C sequence variant has not been reported previously and screening of 135 unrelated healthy individuals for this variant was negative. The substituted amino-acid is located within a region of the SDHD protein frequently altered due to germline mutations in paraganglioma (PGL) families (loss of Y93 [22] and two missense mutations, i.e. D92Y [19,28,46] and L95P [28]). These residues are part of the third transmembrane helix of the SDHD protein [54]. The second mutation has not been reported either. This mutation results from a 4 bp deletion in the 3' exon-intron boundary of exon 3 resulting in skipping of exon 3 leading to a transcript with a premature stop codon. The predicted truncated protein has another carboxyterminal amino-acid sequence from H56 on and its normal function is assumed to be impaired as carboxyterminal amino-acids involved in ubiquinone and heme b binding are missing (H71, D82 and Y83) and consequently the structure of the transmembrane subunit and/or association of the catalytic domain subunits SDHA and SDHB to the membrane would be disrupted [54]. The functional consequence of one sequence variant located within an intronic sequence (IVS2+37ins(TCTA)) is more difficult to evaluate due to lack of fresh tumour material, and parental DNA. Further analysis is needed in order to reveal a possible effect on splicing or RNA stability. Finally, one new and 4 known polymorphisms were detected in 5 NB cell lines and 3 tumour samples. Additional screening for homozygous deletions in all cell lines and methylation in cell lines and tumours were negative. Based upon these results, we can exclude a role for SDHD as a classical tumour suppressor gene in NB. However, the finding of two apparently bona fide SDHD mutations in NB without allelic loss of distal 11q leaves the possibility open that the gene contributes to NB oncogenesis due to haplo-insufficiency, rather than functional inactivation of both alleles. In order to investigate this possibility, we decided to perform further studies at transcript and protein level. Interestingly, SDHD expression was shown to be consistently lower in cell lines with 11q allelic loss versus NB cell lines without loss and also significantly decreased in NB cell lines as compared to normal foetal adrenal neuroblast cells of 16, 18 and 19 weeks gestational time with a mean fold difference of 3.61 between neuroblast cells and NB cell lines. A similar correlation between 11q LOH and reduced SDHD expression was recently described in colorectal and gastric cancer [55]. Our findings at mRNA transcript level, however, did not match with results obtained from further analysis at protein level. Complex II activity and quantitative protein analysis revealed no significant difference between cell lines with or without 11q allelic loss or SDHD mutation. However, measurement of complex II activity might only reflect part of the functional properties of SDHD. Also, measurement of differences in protein quantity is far less sensitive than Q-PCR at transcript levels. Therefore, these observations at present do not fully exclude SDHD involvement in NB. Finally, we also looked at the morphologic characteristics of the mitochondria as a possible clue to SDHD dysfunction. In keeping with, at best, partial loss of function of SDHD, we did not observe similar gross morphologic changes as reported for PGL with SDHD mutations (swelling with loss of matrix density and generalized rarefaction of cristae), the latter being characterized by destabilization of complex II with loss of enzymatic activity [48]. However, most of the cell lines showed dilated mitochondrial cristae. It has been demonstrated that this is a reversible phenomenon, and parallels arise in intracellular ADP/ATP ratio or low energy state [56]. Subsequent combined ultrastructural and biochemical studies from several authors indicated that dilation of cristae follows a decrease in mitochondrial membrane potential that can be provoked by various experimental procedures [57,58]. This configuration was detected in only a small percentage of mitochondria in archived sections of NB tumours and in sections published earlier. Morphologic analysis of mitochondria in NB thus far received little attention. The true significance of the observed mitochondrial morphological changes in NB is intriguing, but does not appear to be related to the mutations we have found. Conclusions In contrast to previous findings in PGL and PC, this study excludes a classical two hit Knudson model for SDHD involvement in NB. However, the finding of, albeit rare, bona fide mutations and reduced expression of SDHD in NB with 11q allelic loss hints at a possible haplo-insufficient contribution to tumour development. A better understanding of the different functions of SDHD, in particular its possible contribution to energy independent apoptosis involving the release of cytochrome c and procaspases, will allow further functional assays to asses how this gene contributes to tumour development in general, and the high stage NB phenotype in particular [59,60]. Evidence for contribution to a cancer phenotype through haplo-insufficiency has recently been obtained for a number of loci, including CDKN1B (p27Kip1) [61,62], TP53 (p53) [63], DMP1 [64], PTEN [65], APC [66] and NKX3.1 [67]. In mouse models for some of these genes, loss or mutation of one allele increased tumour susceptibility despite expression of the remaining wild-type allele [68]. Although the present data on protein and functional level do not provide consistent evidence for the haplo-insufficient involvement of SDHD in NB, a bipartite mechanism as tumour suppressor gene for the SDHD gene, as described for the APC gene can at present not be fully excluded. Following this hypothesis, germline mutations in SDHD would predispose to PGL or PC development. Rare somatic mutations and more typically loss of one allele could contribute to the metastasizing NB tumour phenotype (and possible also other tumour types), not as an initiating step but rather as later event in tumour development. However, further evidence is needed to support the haplo-insufficient involvement of SDHD in cancer. Ultimately, knockout mice for the SDHD gene leading to haplo-insufficiency for SDHD in neuroblast progenitor cells, would be the appropriate test to evaluate this hypothesis. List of abbreviations AIF = allelic imbalance factor CGH = comparative genomic hybridization DHPLC = denaturing high performance liquid chromatography LOH = loss of heterozygosity MGB = minor groove binder MSP = methylation specific PCR NB = neuroblastoma PC = pheochromocytoma PGL = paraganglioma ROS = reactive oxygen species SDHD = succinate dehydrogenase, subunit D SNP = single nucleotide polymorphism SRO = shortest region of overlap Competing interests The authors declare that they have no competing interests. Author's contributions KDP carried out the genomic and transcriptomic studies, and drafted the manuscript. JH performed the methylation studies. JS carried out the immunoblottings and spectrophotometric analysis that was evaluated by RVC. AN performed the ultrastructural analysis that was screened and discussed by CV, FR and MP. GL and NVR collected the tumour material. JV and FS participated in the study's design and coordination. All authors have reviewed the manuscript and FS and ADP were the final editors of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We would like to thank E. George for the spectrophotometrical assays, G. De Vos and P. Degraeve for the cell cultures and Petra Van Acker and Inge Vereecke for their help with the DHPLC analyses. This text presents research results of the Belgian program of Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The scientific responsibility is assumed by the authors. This work was supported by BOF-grant 011F1200 and 011B4300, GOA-grant 12051203 and FWO-grant G.0028.00. Katleen De Preter is an aspirant with the Fund for Scientific Research, Flanders (FWO-Vlaanderen). JV is supported by a post-doctoral grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). Nadine Van Roy is a postdoctoral researcher with the FWO. Figures and Tables Figure 1 Details of sequencing profiles: (A) Deletion of GGCA in cell line N206 causing skip of exon 3 and (B) Y93C mutation in cell line NMB; (C) RT-PCR (reverse transcriptase PCR) on cell lines grown with or without puromycine (T = treated, U = untreated) revealed a transcript variant in cell line N206, caused by the GGCA deletion (lane 1 and 2). Figure 2 SDHD mRNA levels in NB cell lines (light gray), neuroblast cells (gray) and human normal control samples (white). Significantly reduced SDHD mRNA expression levels in NB cell lines with 11q23 allelic loss compared to 11q23 intact NB cell lines (P = 5.31E-06) and normal tissue samples (P = 1.49E-05). Figure 3 Mitochondrial ultrastructure shows heterogeneity between cell lines (same final magnification for the 4 images, marker = 0.5 μm): (A) NB cell line N206: dilated crista spaces in small mitochondria with a dense matrix; (B) NB cell line NMB: small mitochondria with narrow cristae and light matrix, so-called orthodox configuration, (C) NB cell line SJNB-8: unusually large mitochondria in orthodox configuration (narrow cristae), some areas in the matrix are cleared and lack cristae; (D) NB cell line LA-N-2: very large mitochondria with dilated cristae and dense matrix. Table 1 Primer sequences used for homozygous exon deletion screening with real-time quantitative PCR amplicon forward reverse SDHD exon 1 5'AGGAACGAGATGGCGGTTCT3' 5'TCCTAGGGCACCGCAAAC3' SDHD exon 2 5'CCAGTGGTCAGACCTGCTCAT3' 5'TGCTGCACTCCACACCATTC3' SDHD exon 3 5'GACTAGCGAGAGGGTTGTCAGTGT3' 5'CATCGCAGAGCAAGGATTCA3' SDHD exon 4 5'TGGCACTTTCAGCTTTAACCTTT3' 5'CACAGCATGGCAACAGCTTT3' Table 2 Primer sequences used for denaturing high performance liquid chromatography (DHPLC) and sequencing amplicon forward reverse SDHD exon 1 5'GCACCGCCTCTCGACTTC3' 5'TGCTGTGATTTCGGTATTTTCTTC3' SDHD exon 2 5'AACCCCAGTGAAATAGATGCTATCTTC3' 5'AGTCCTGCTAAAGGCATGACCATTA3' SDHD exon 3 5'CACTGCCTGTCAGTTTGGGTTAC3' 5'GGGCATTTCAATCAACTTCTCCC3' SDHD exon 4 5'TCCCCTAAAGAAGCAAACAGTGAC3' 5'GAGCTTAATGGCATGACAAAGCAG3' SDHD exon 4 Noncoding region (only for sequencing) 5'GTGGTTTTTTATTGATGTTATGATTTT3' 5'AATCTCAATTTACAGTTGGTAGTATTTT3' Table 3 (A) 11q23 LOH data of NB patients (35 patients form the Molecular Oncology Unit (Lyon, France) (F-samples) and 32 patients from the Ghent University Hospital (Ghent, Belgium) (G-samples)). Based on the allelic imbalance factor (AIF) of 6 markers (4 on 11q23 and 2 on 11p) normal 11q23 status was distinguished from unbalanced 11q LOH and loss of both 11q and 11p (here indicated as whole chromosome loss) (- = no data available) and (B) 11q23 status in NB cell lines based on FISH, LOH, karyotypes, CGH and/or M-FISH A NB tumour case 11q23 LOH status MYCN ampl 1p del stage NB tumour case 11q23 LOH status MYCN ampl 1p del stage F1 normal no no 3 G1 normal no no 4 F2 normal no no 3 G2 whole chr11 loss no no 4 F3 normal no no 3 G3 normal no no 1 F4 normal no no 4 G4 normal no no 1 F5 whole chr11 loss no no 4 G5 normal no no 2 F6 unb [11q]LOH no no 4 G6 normal - no 3 F7 normal no no 4 G7 normal - - 4 F8 normal no no 3 G8 normal no no 1 F9 normal no no 4 G9 whole chr11 loss no no 4 F10 whole chr11 loss no no 3 G10 normal no no 4 F11 normal no no 4 G11 normal no no 1 F12 normal no no 4 G12 normal no no 4 F13 normal no no 3 G13 normal no yes 1 F14 whole chr11 loss no no 3 G14 whole chr11 loss no no 3 F15 whole chr11 loss no yes 4 G15 normal no no 3 F16 normal no no 3 G16 normal - no 4 F17 unb [11q]LOH no no 4 G17 normal no no 3 F18 unb [11q]LOH no yes 4 G18 normal no yes 1 F19 normal no no 4 G19 normal no no 4S F20 normal no no 3 G20 normal yes yes 3 F21 unb [11q]LOH no no 4 G21 unb [11q]LOH no no 3 F22 whole chr11 loss no no 3 G22 unb [11q]LOH yes yes 4 F23 normal no no 3 G23 normal no no 4 F24 normal no no 3 G24 normal yes yes 4 F25 whole chr11 loss no no 3 G25 normal no no 4 F26 unb [11q]LOH no no 4 G26 normal no yes 4 F27 whole chr11 loss no no 3 G27 normal no no 3 F28 normal no no 3 G28 normal - no 2 F29 unb [11q]LOH no no 4 G29 normal no no 3 F30 normal no yes 4 G30 normal no no 1 F31 normal no no 4 G31 normal - no 3 F32 normal no no 4 G32 normal no no 4S F33 whole chr11 loss no no 3 F34 normal no yes 4 F35 unb [11q]LOH no no 4 B NB cell line 11q23 status MYCN ampl 1p del CHP-134 normal yes yes CHP-901 normal yes yes CHP-902R normal yes yes CLB-GA deletion no no GI-M-EN deletion no yes IMR-32 deletion yes yes LA-N-1 normal yes yes LA-N-2 normal yes no LA-N-5 normal yes yes LA-N-6 deletion no yes N206 normal yes yes NBL-S deletion no no NGP deletion yes yes NLF normal yes yes NMB normal yes yes SJNB-12 normal no yes SJNB-1 normal no yes SJNB-10 normal yes yes SJNB-6 normal yes yes SJNB-8 normal yes yes SK-N-AS deletion no yes SK-N-BE normal yes yes SK-N-FI normal no no SK-N-SH normal no no SMS-KAN normal yes yes SMS-KCNR normal yes yes STA-NB-10 normal yes yes STA-NB-3 normal yes yes STA-NB-8 normal yes yes TR-14 normal yes yes UHG-NP normal yes yes Table 4 SDHD base pair variants found in NB tumour samples and cell lines, the position of the variant, the change in the protein caused by the variant and the conclusion (mutation or polymorphism); also listed for each tumour and cell line are the genomic status for chromosome arm 11q (n.d. = not done) (for cell lines: chromosome 11 centromere copy number versus 11q23 copy number according to FISH, for tumours: normal, unbalanced LOH (unb [11q]LOH) or whole chromosome 11 loss according to microsatellite marker analysis), MYCN status (normal or amplified), 1p status and tumour stage when available (- = not available). NB tumour case number / NB cell line 11q23 status by microsatellite marker analysis 11q23 status by FISH SDHD base pair variant exon change in protein mutation (M) or polymorphism (P) variant present in constitutional (C)/ tumour (T) MYCN 1p del stage F11 normal n.d. g.6911ins(TCTA) IVS2+37ins(TCTA) ? (see text) ? (see text) C+T normal no 4 F18 unb [11q] LOH n.d. g.5842 G>A 1 G12S P C+T normal yes 4 g.7750 A>G IVS3-29 A>G P C+T g.7802 C>T 3 S68S P C+T g.13678 T>C IVS4-32 T>C P C+T F22 whole chr11 loss n.d. g.7750 A>G IVS3-29 A>G P C normal no 3 g.7802 C>T 3 S68S P C g.13678 T>C IVS4-32 T>C P C F35 unb [11q] LOH n.d. g.7750 A>G IVS3-29 A>G P C+T g.7802 C>T 3 S68S P C+T g.13678 T>C IVS4-32 T>C P C+T LA-N-2 n.d. 2/2 heterozygous g.6854 A>G 2 H50R P - amplified no 4 N206 n.d. 2/2 heterozygous g.79124del(GGCA) 3 exon 3 skip, premature stop codon M - amplified yes 4 NGP n.d. 2/1 hemizygous g.7750 A>G IVS3-29 A>G P - amplified yes - g.7802 C>T 3 S68S P g.13678 T>C IVS4-32 T>C P NMB n.d. 4/3 allelic imbalance/ heterozygous (based on sequence) g.7750 A>G IVS3-29 A>G P - amplified yes 4 g.7802 C>T 3 S68S P g.7876 A>G 3 Y93C M g.13678 T>C IVS4-32 T>C P SK-N-FI n.d. 2/2 heterozygous g.7750 A>G IVS3-29 A>G P - no no - g.7802 C>T 3 S68S P g.13678 T>C IVS4-32 T>C P Table 5 Ultrastructural analysis of mitochondria dilation of cristae* density of matrix° configuration# matrix granules°° mean projected length (μm) max projected length (μm) CHP-901 + -+ + + 0,65 1,31 CLB-GA ++ + + 0,69 1,92 IMR-32 + + + - 0,69 1,27 LA-N-2 ++ + - 0,82 2,18 MCF-7 - + + - 1,06 3,11 N-206 +++ + - 0,84 1,65 NGP +++ + - 0,84 1,74 NMB - - + - 0,68 1,26 SJNB-10 +++ + - 0,63 1,69 SJNB-12 + + - 0,47 1,23 SJNB-8 - -+ + - 1,02 3,40 SK-N-AS ++ + - 0,66 1,47 SK-N-FI + - + - 0,65 1,32 SK-N-MC - - + - 0,90 2,03 SK-N-SH + -+ + + 0,56 1,31 *: +cristae of some mitochondria dilated; ++cristae of many mitochondria dilated; +++all mitochondria have dilated cristae; °: +matrix of most mitochondria is more electron dense than the cytosol; -+part of the mitochondria are dense, but others have a light matrix; -mitochondrial matrix is lighter than or equal to cytosol; #: +mitochondria with an orthodox configuration are present; in the same cell or culture other mitochondria may have dilated cristae and a dense matrix; °°: -few or no matrix granules; +matrix granules visible in many or most mitochondria, normal image; ==== Refs Cooper MJ Hutchins GM Cohen PS Helman LJ Mennie RJ Israel MA Human neuroblastoma tumor cell lines correspond to the arrested differentiation of chromaffin adrenal medullary neuroblasts Cell Growth Differ 1990 1 149 59 1982060 Bown N Cotterill S Lastowska M O'Neill S Pearson AD Plantaz D Meddeb M Danglot G Brinkschmidt C Christiansen H Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma N Engl J Med 1999 340 1954 61 10379019 10.1056/NEJM199906243402504 Maris JM Matthay KK Molecular biology of neuroblastoma J Clin Oncol 1999 17 2264 79 10561284 Brodeur GM Neuroblastoma: biological insights into a clinical enigma Nat Rev Cancer 2003 3 203 16 12612655 10.1038/nrc1014 Plantaz D Vandesompele J Van Roy N Lastowska M Bown N Combaret V Favrot MC Delattre O Michon J Benard J Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification Int J Cancer 2001 91 680 6 11267980 10.1002/1097-0215(200002)9999:9999<::AID-IJC1114>3.0.CO;2-R Vandesompele J Van Roy N Van Gele M Laureys G Ambros P Heimann P Devalck C Schuuring E Brock P Otten J Genetic heterogeneity of neuroblastoma studied by comparative genomic hybridization Genes Chromosomes Cancer 1998 23 141 52 9739017 10.1002/(SICI)1098-2264(199810)23:2<141::AID-GCC7>3.0.CO;2-2 Vandesompele J Speleman F Van Roy N Laureys G Brinskchmidt C Christiansen H Lampert F Lastowska M Bown N Pearson A Multicentre analysis of patterns of DNA gains and losses in 204 neuroblastoma tumors: how many genetic subgroups are there? Med Pediatr Oncol 2001 36 5 10 11464905 10.1002/1096-911X(20010101)36:1<5::AID-MPO1003>3.0.CO;2-E Luttikhuis ME Powell JE Rees SA Genus T Chughtai S Ramani P Mann JR McConville CM Neuroblastomas with chromosome 11q loss and single copy MYCN comprise a biologically distinct group of tumours with adverse prognosis Br J Cancer 2001 85 531 7 11506492 10.1054/bjoc.2001.1960 Breen CJ O'Meara A McDermott M Mullarkey M Stallings RL Coordinate deletion of chromosome 3p and 11q in neuroblastoma detected by comparative genomic hybridization Cancer Genet Cytogenet 2000 120 44 9 10913676 10.1016/S0165-4608(99)00252-6 Srivatsan ES Murali V Seeger RC Loss of heterozygosity for alleles on chromosomes 11q and 14q in neuroblastoma Prog Clin Biol Res 1991 366 91 8 2068183 Maris JM Guo C White PS Hogarty MD Thompson PM Stram DO Gerbing R Matthay KK Seeger RC Brodeur GM Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma Med Pediatr Oncol 2001 36 24 7 11464895 10.1002/1096-911X(20010101)36:1<24::AID-MPO1007>3.0.CO;2-7 Vandesompele J Baudis M De Preter K Van Roy N Ambros P Bown N Combaret V Lastowska M Nicholson JC O'Meara A Unequivocal delineation of clinico-genetic subgroups and identification of a long term survivor signature for neuroblastoma J Clin Oncol Spitz R Hero B Ernestus K Berthold F Deletions in chromosome arms 3p and 11q are new prognostic markers in localized and 4s neuroblastoma Clin Cancer Res 2003 9 52 8 12538451 Bader SA Fasching C Brodeur GM Stanbridge EJ Dissociation of suppression of tumorigenicity and differentiation in vitro effected by transfer of single human chromosomes into human neuroblastoma cells Cell Growth Differ 1991 2 245 55 1679663 Guo C White PS Hogarty MD Brodeur GM Gerbing R Stram DO Maris JM Deletion of 11q23 is a frequent event in the evolution of MYCN single-copy high-risk neuroblastomas Med Pediatr Oncol 2000 35 544 6 11107113 10.1002/1096-911X(20001201)35:6<544::AID-MPO10>3.0.CO;2-2 Guo C White PS Weiss MJ Hogarty MD Thompson PM Stram DO Gerbing R Matthay KK Seeger RC Brodeur GM Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas Oncogene 1999 18 4948 57 10490829 10.1038/sj.onc.1202887 Hirawake H Taniwaki M Tamura A Amino H Tomitsuka E Kita K Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase Biochim Biophys Acta 1999 1412 295 300 10482792 10.1016/S0005-2728(99)00071-7 Hirawake H Taniwaki M Tamura A Kojima S Kita K Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23 Cytogenet Cell Genet 1997 79 132 8 9533030 Baysal BE Ferrell RE Willett-Brozick JE Lawrence EC Myssiorek D Bosch A van der Mey A Taschner PE Rubinstein WS Myers EN Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma Science 2000 287 848 51 10657297 10.1126/science.287.5454.848 Neumann HP Bausch B McWhinney SR Bender BU Gimm O Franke G Schipper J Klisch J Altehoefer C Zerres K Germ-line mutations in nonsyndromic pheochromocytoma N Engl J Med 2002 346 1459 66 12000816 10.1056/NEJMoa020152 Gimm O Armanios M Dziema H Neumann HP Eng C Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma Cancer Res 2000 60 6822 5 11156372 Badenhop RF Cherian S Lord RS Baysal BE Taschner PE Schofield PR Novel mutations in the SDHD gene in pedigrees with familial carotid body paraganglioma and sensorineural hearing loss Genes Chromosomes Cancer 2001 31 255 63 11391796 10.1002/gcc.1142 Gimenez-Roqueplo AP Favier J Rustin P Mourad JJ Plouin PF Corvol P Rotig A Jeunemaitre X The R22X mutation of the SDHD gene in hereditary paraganglioma abolishes the enzymatic activity of complex II in the mitochondrial respiratory chain and activates the hypoxia pathway Am J Hum Genet 2001 69 1186 97 11605159 10.1086/324413 Baysal BE Willett-Brozick JE Lawrence EC Drovdlic CM Savul SA McLeod DR Yee HA Brackmann DE Slattery WH 3rdMyers EN Prevalence of SDHB, SDHC, and SDHD germline mutations in clinic patients with head and neck paragangliomas J Med Genet 2002 39 178 83 11897817 10.1136/jmg.39.3.178 Aguiar RC Cox G Pomeroy SL Dahia PL Analysis of the SDHD gene, the susceptibility gene for familial paraganglioma syndrome (PGL1), in pheochromocytomas J Clin Endocrinol Metab 2001 86 2890 4 11397905 10.1210/jc.86.6.2890 Masuoka J Brandner S Paulus W Soffer D Vital A Chimelli L Jouvet A Yonekawa Y Kleihues P Ohgaki H Germline SDHD mutation in paraganglioma of the spinal cord Oncogene 2001 20 5084 6 11526495 10.1038/sj.onc.1204579 Kytola S Nord B Elder EE Carling T Kjellman M Cedermark B Juhlin C Hoog A Isola J Larsson C Alterations of the SDHD gene locus in midgut carcinoids, Merkel cell carcinomas, pheochromocytomas, and abdominal paragangliomas Genes Chromosomes Cancer 2002 34 325 32 12007193 10.1002/gcc.10081 Taschner PE Jansen JC Baysal BE Bosch A Rosenberg EH Brocker-Vriends AH van Der Mey AG van Ommen GJ Cornelisse CJ Devilee P Nearly all hereditary paragangliomas in the Netherlands are caused by two founder mutations in the SDHD gene Genes Chromosomes Cancer 2001 31 274 81 11391798 10.1002/gcc.1144 Jogi A Ora I Nilsson H Lindeheim A Makino Y Poellinger L Axelson H Pahlman S Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype Proc Natl Acad Sci U S A 2002 99 7021 6 12011461 10.1073/pnas.102660199 Schleiermacher G Janoueix-Lerosey I Combaret V Derre J Couturier J Aurias A Delattre O Combined 24-color karyotyping and comparative genomic hybridization analysis indicates predominant rearrangements of early replicating chromosome regions in neuroblastoma Cancer Genet Cytogenet 2003 141 32 42 12581896 10.1016/S0165-4608(02)00644-1 Van Roy N Van Limbergen H Vandesompele J Van Gele M Poppe B Salwen H Laureys G Manoel N De Paepe A Speleman F Combined M-FISH and CGH analysis allows comprehensive description of genetic alterations in neuroblastoma cell lines Genes Chromosomes Cancer 2001 32 126 35 11550280 10.1002/gcc.1174 Van Roy N Jauch A Van Gele M Laureys G Versteeg R De Paepe A Cremer T Speleman F Comparative genomic hybridization analysis of human neuroblastomas: detection of distal 1p deletions and further molecular genetic characterization of neuroblastoma cell lines Cancer Genet Cytogenet 1997 97 135 42 9283597 10.1016/S0165-4608(96)00362-7 De Preter K Speleman F Combaret V Lunec J Laureys G Eussen BH Francotte N Board J Pearson AD De Paepe A Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay Mod Pathol 2002 15 159 66 11850545 10.1038/modpathol.3880508 Van Roy N Laureys G Cheng NC Willem P Opdenakker G Versteeg R Speleman F 1;17 translocations and other chromosome 17 rearrangements in human primary neuroblastoma tumors and cell lines Genes Chromosomes Cancer 1994 10 103 14 7520263 Devilee P Cleton-Jansen AM Cornelisse CJ Ever since Knudson Trends Genet 2001 17 569 73 11585662 10.1016/S0168-9525(01)02416-7 Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands Proc Natl Acad Sci U S A 1996 93 9821 6 8790415 10.1073/pnas.93.18.9821 Li LC Dahiya R MethPrimer: designing primers for methylation PCRs Bioinformatics 2002 18 1427 31 12424112 10.1093/bioinformatics/18.11.1427 Vandesompele J De Paepe A Speleman F Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR Anal Biochem 2002 303 95 8 11906156 10.1006/abio.2001.5564 De Preter K Vandesompele J Heimann P Kockx MM Van Gele M Hoebeeck J De Smet E Demarche M Laureys G Van Roy N Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells Cancer Lett 2003 197 53 61 12880960 10.1016/S0304-3835(03)00084-3 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes Genome Biol 2002 3:RESEARCH0034 1 11 Rustin P Chretien D Bourgeron T Gerard B Rotig A Saudubray JM Munnich A Biochemical and molecular investigations in respiratory chain deficiencies Clin Chim Acta 1994 228 35 51 7955428 10.1016/0009-8981(94)90055-8 Van Coster R Smet J George E De Meirleir L Seneca S Van Hove J Sebire G Verhelst H De Bleecker J Van Vlem B Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects Pediatr Res 2001 50 658 65 11641463 Cascon A Ruiz-Llorente S Cebrian A Leton R Telleria D Benitez J Robledo M G12S and H50R variations are polymorphisms in the SDHD gene Genes Chromosomes Cancer 2003 37 220 221 12696072 10.1002/gcc.10212 Cascon A Ruiz-Llorente S Cebrian A Telleria D Rivero JC Diez JJ Lopez-Ibarra PJ Jaunsolo MA Benitez J Robledo M Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma Eur J Hum Genet 2002 10 457 61 12111639 10.1038/sj.ejhg.5200829 Lima J Maximo V Soares P Sobrinho-Simoes M Alterations of the SDHD gene locus in midgut carcinoids Genes Chromosomes Cancer 2003 36 424 12619155 10.1002/gcc.10147 Dannenberg H Dinjens WN Abbou M Van Urk H Pauw BK Mouwen D Mooi WJ de Krijger RR Frequent germ-line succinate dehydrogenase subunit D gene mutations in patients with apparently sporadic parasympathetic paraganglioma Clin Cancer Res 2002 8 2061 6 12114404 Hui AB Lo KW Chan SY Kwong J Chan AS Huang DP Absence of SDHD mutations in primary nasopharyngeal carcinomas Int J Cancer 2002 97 875 7 11857371 10.1002/ijc.10066 Douwes Dekker PB Hogendoorn PC Kuipers-Dijkshoorn N Prins FA van Duinen SG Taschner PE van der Mey AG Cornelisse CJ SDHD mutations in head and neck paragangliomas result in destabilization of complex II in the mitochondrial respiratory chain with loss of enzymatic activity and abnormal mitochondrial morphology J Pathol 2003 201 480 6 14595761 10.1002/path.1461 Takahashi H Ikuta F Tsuchida T Tanaka R Ultrastructural alterations of neuronal cells in a brain stem ganglioglioma Acta Neuropathol (Berl) 1987 74 307 12 3673523 Taxy JB Electron microscopy in the diagnosis of neuroblastoma Arch Pathol Lab Med 1980 104 355 60 6893122 Staley NA Poleksy HF Bensch KG Fine structural and biochemical studies on the malignant ganglioneuroma J Neuropathol Exp Neurol 1967 26 634 53 6053734 Mackay B Masse SR King OY Butler J Diagnosis of neuroblastoma by electron microscopy of bone marrow aspirates Pediatrics 1975 56 1045 9 1196755 Misugi K Misugi N Newton WA Jr Fine structural study of neuroblastoma, ganglioneuroblastoma, and pheochromocytoma Arch Pathol 1968 86 160 70 5660622 Yankovskaya V Horsefield R Tornroth S Luna-Chavez C Miyoshi H Leger C Byrne B Cecchini G Iwata S Architecture of succinate dehydrogenase and reactive oxygen species generation Science 2003 299 700 4 12560550 10.1126/science.1079605 Habano W Sugai T Nakamura S Uesugi N Higuchi T Terashima M Horiuchi S Reduced expression and loss of heterozygosity of the SDHD gene in colorectal and gastric cancer Oncol Rep 2003 10 1375 80 12883710 Hackenbrock CR Rehn TG Weinbach EC Lemasters JJ Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell J Cell Biol 1971 51 123 37 5111873 10.1083/jcb.51.1.123 Ord MJ Smith RA Correlation of mitochondrial form and function in vivo: microinjection of substrate and nucleotides Cell Tissue Res 1982 227 129 37 7172205 10.1007/BF00206336 Gottlieb E Armour SM Harris MH Thompson CB Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis Cell Death Differ 2003 10 709 17 12761579 10.1038/sj.cdd.4401231 van Loo G Saelens X van Gurp M MacFarlane M Martin SJ Vandenabeele P The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet Cell Death Differ 2002 9 1031 42 12232790 10.1038/sj.cdd.4401088 Ravagnan L Roumier T Kroemer G Mitochondria, the killer organelles and their weapons J Cell Physiol 2002 192 131 7 12115719 10.1002/jcp.10111 Pietenpol JA Bohlander SK Sato Y Papadopoulos N Liu B Friedman C Trask BJ Roberts JM Kinzler KW Rowley JD Assignment of the human p27Kip1 gene to 12p13 and its analysis in leukemias Cancer Res 1995 55 1206 10 7882309 Fero ML Randel E Gurley KE Roberts JM Kemp CJ The murine gene p27Kip1 is haplo-insufficient for tumour suppression Nature 1998 396 177 80 9823898 10.1038/24179 Venkatachalam S Shi YP Jones SN Vogel H Bradley A Pinkel D Donehower LA Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation Embo J 1998 17 4657 67 9707425 10.1093/emboj/17.16.4657 Inoue K Zindy F Randle DH Rehg JE Sherr CJ Dmp1 is haplo-insufficient for tumor suppression and modifies the frequencies of Arf and p53 mutations in Myc-induced lymphomas Genes Dev 2001 15 2934 9 11711428 10.1101/gad.929901 Kwabi-Addo B Giri D Schmidt K Podsypanina K Parsons R Greenberg N Ittmann M Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression Proc Natl Acad Sci U S A 2001 98 11563 8 11553783 10.1073/pnas.201167798 Yan H Dobbie Z Gruber SB Markowitz S Romans K Giardiello FM Kinzler KW Vogelstein B Small changes in expression affect predisposition to tumorigenesis Nat Genet 2002 30 25 6 11743581 10.1038/ng799 Magee JA Abdulkadir SA Milbrandt J Haploinsufficiency at the Nkx3.1 locus. A paradigm for stochastic, dosage-sensitive gene regulation during tumor initiation Cancer Cell 2003 3 273 83 12676585 10.1016/S1535-6108(03)00047-3 Quon KC Berns A Haplo-insufficiency? Let me count the ways Genes Dev 2001 15 2917 21 11711426 10.1101/gad.949001

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==== Front BMC Musculoskelet DisordBMC Musculoskeletal Disorders1471-2474BioMed Central London 1471-2474-5-291532915010.1186/1471-2474-5-29Research ArticleOsteoporosis-related life habits and knowledge about osteoporosis among women in El Salvador: A cross-sectional study Hernandez-Rauda Roberto [email protected] Sandra [email protected] Facultad de Ciencias de la Salud, Universidad Andrés Bello, 1a Calle Poniente y 41a Avenida Norte, 2128, Colonia Flor Blanca, San Salvador, El Salvador, América Central2004 26 8 2004 5 29 29 31 12 2003 26 8 2004 Copyright © 2004 Hernandez-Rauda and Martinez-Garcia; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Osteoporosis is a systemic skeletal disorder, characterized by reduced bone mass, deterioration of bone structure, increased bone fragility, and increased fracture risk. It is more frequent to find among women than men at a 4:1 ratio. Evidence suggests that to adopt changes on some life habits can prevent or delay development of osteoporosis. Several osteoporosis-risk factors have been confirmed in the US and western Europe, but in El Salvador there are neither reliable epidemiological statistics about this skeletal disorder nor studies addressing osteoporosis-risk factors in women. The aim of this study was to determinate the extent of osteoporosis knowledge, the levels of both daily calcium intake and weight-bearing physical activity, and the influence of several osteoporosis-risk factors on these variables in three age groups of Salvadorean women. Methods In this exploratory cross-sectional study, an osteoporosis knowledge assessment questionnaire incluiding a food frequency and a physical activity record section were used to collect data and it was delivered through a face-to-face interview. A convenience sample (n = 197) comprised of three groups of women aged 25–35 years, 36–49 years, and over 49 years was taken. Among-group comparisons of means were analyzed by two-way ANOVA. To determinate the overall influence of osteoporosis-risk factors, the multivariate analysis was used. Results Study results indicated that better educated women had more knowledge about osteoporosis than women with a low education level, regardless of age, even though this knowledge was rather fair. Older women got more weight-bearing physical activity at home and less at place of employment than reported by the younger women; however, neither group performed sufficient high-intensity WBPA to improve bone mass. Regardless of age, the most women consumed 60% or less than the Dietary Reference Intake of calcium and depend on household income, lactose intolerance and coffee rather than milk consumption. Conclusion In summary, the majority of women in this study have modest knowledge on osteoporosis. The knowledge base is not linked to preventive health habits, including sufficient calcium intake and performance of weight-bearing physical activities. They are thus at increased risk for low bone mass. ==== Body Background Osteoporosis is a systemic skeletal disorder, characterized by reduction of bone mass, deterioration of bone structure, increasing bone fragility, and increasing fracture risk [1-4]. It is more frequent among women than among men [5,6]. The development of low bone mass is typically asymptomatic, with many women reporting clinical manifestations including acute back pain, limited back mobility, fragility fractures (hip, vertebrae, proximal femur, distal radius, humerus, tibia), compression of midthoracic vertebrae and upper lumbar vertebrae, progressive deformation of the spinal column (cyphosis), reduced height, and radiculopathies [2,4-6,8,9]. Several risk factors for osteoporosis have been identified, these include female sex; Caucasian or Asiatic race; advancing age; family history of osteoporosis or fragility fractures; a low body mass index; menopause before age 45 years; prolonged amenorrhea unrelated to menopause; nulliparity; prolonged lactation; diet low in calcium and vitamin D; poor intestinal absorption of calcium; lactose intolerance; excessive caffeine or alcohol consumption; smoking; sedentary lifestyle; and prolonged treatment with thyroid hormones, glucocorticoids (e.g. cortisone), anticonvulsants, aluminum antiacids, and use of anticoagulants [1,3-6,8]. Approximately 20% of bone mass is genetically determined; however, the risk of osteoporosis can be reduced by optimizing bone mass increasing during youth, conserving bone mass during adulthood, and minimizing bone mass loss during advancing age [5-7]. Among most important preventive habits are a) weight-bearing exercise (e.g. going up and down stairs, jogging, aerobics, swimming, and isometrics; at least 30 minutes daily), b) diet or supplements containing adequate levels of calcium and vitamin D, and c) absence or cessation of smoking and no greater than moderate alcohol and/or caffeine consumption [5-10]. A study on US women aged over 25 years found that knowledge about osteoporosis was limited, irrespective of age [11]. Calcium intake was sufficient in most cases, but the amount and type of physical activity was inadequate to achieve enhanced bone mass; most women in this study performed some physical activity in the course of paid work or housework, but did not admit to systematic daily exercise. Other study on Caucasian and African-American women found that most of them had heard about osteoporosis, but few women got both adequate exercise and the recommended intake of calcium per day [12]. Asian women in Australia also had lower calcium intake (< 800 mg/day) and their knowledge about osteoporosis was limited [13]. There have been three related studies reported in Hispanic women: a study performed in Mexico [14], and two studies on women of Hispanic origin in the US [15,16]. The Mexican study considered women aged 50 – 59 years, and focused on knowledge about menopause and risk associated with premature menopause. About 90% of subjects were aware of the relationship between menopause and osteoporosis, but most subjects had little knowledge of other risk factors, and incorporated life habits that clearly increased osteoporosis risk. The studies on women of Hispanic origin in the US have yielded somewhat contradictory results. The first of these found that more than 37% of women had adequate preventive habits including the taking of calcium supplements and the performance of regular exercise; much of this was attributed to prior health education, knowledge about osteoporosis, bone-mass evaluation offered by healthcare services, and medical advice [15]. The second study considered both Hispanic and African-American women, and found that most women in both groups had a poor knowledge of behaviors that promote and maintain bone mass [16]. Notably, less than 50% of women performed regular physical exercise, and less than 10% had adequate calcium intake. At present, the Salvadorean Public Health and Social Assistance Ministry does not maintain a specific record of this disease in adults, and most cases are likely classified as dorsalgy, that includes several musculoskeletal diseases such as radiculopathies, cervicalgy, lumbalgy, sciatica, spinal derived pain, and unspecified back pain [17]. This reflects the fact that osteoporosis may be reported as dorsalgy. According to this report from El Salvador, in 2001 dorsalgy was the seventh most frequent cause of morbidity in women aged 50 – 59 years attending outpatient clinics, with an incidence of 3,983 new cases per 100,000 inhabitants and a total of 8,989 consultations (first-time or subsequent); in women aged 60 years or more, there were 9,754 consultations for dorsalgy in 2001. Bearing in mind the lack of reliable epidemiological data, the present study wished to investigate osteoporosis-related life habits (including exercise and calcium intake) and knowledge about osteoporosis among Salvadorean women aged ≥ 25 years. We investigated possible relationships of these variables with age, educational level, household income, family history of osteoporosis, menopause before age 45 years, fecundity (children per woman), lactose intolerance, caffeine consumption, low in calcium diet, and use of aluminum antiacids. Methods Study design and sampling This was an exploratory study with a cross-sectional design performed between May and September 2003, and it was used to survey a convenience sample comprised of 197 women (73 aged 25 – 35 years, 74 aged 36 – 49 years, and 50 aged over 49 years) from urban areas within 6 main municipalities in El Salvador. All subjects were randomly sampled and recruited by personal contact through to visit homes, churches, schools, primary healthcare centers, hospitals, supermarkets, shopping centers, and parks. It was not necessary to obtain any proper informed consent from interviewed women. The participation rate of women was 87.5% (197 out of 225). We selected three age groups of women attending following criterions: women aged 25 – 35 years are at the period when higher peak of bone mass is reached [3]; women aged 36 – 49 years are at a period around menopause when bones undergo a slow mineral density loss [3]; finally, most women aged over 49 years have undergone menopause, when osteoporosis clinical manifestations may begin to show [3]. Data collection and instruments Data were obtained at the time of interview, which were performed by a member of the research team. Prior training in interview techniques was obtained for study. Each staff interviewer subsequently participated in several practice sessions, with an evaluation component to confirm accurate transcription of responses, and comparison and adjustment to ensure good inter-interviewer concordance. The data collection instrument incorporated a personal interview guide comprising five sections, validated through a prior pilot study performed in 12 women (4 in each age group). Among the demographic information was household income. It was classified by the criteria of the Salvadorean Economy Ministry as "below poverty line" if monthly income was below the cost of two basic shopping baskets (i.e. < $254), and otherwise as "above poverty line" [18]. The second survey section generated information on family history of osteoporosis, and asked about the use of aluminum antiacids. Section 3 addressed weight-bearing physical activity (e.g. walking, standing, climbing and descending stairs) both at place of employment and at home, and included questions on weight-bearing exercises including jogging, swimming, aerobics, and isometrics. This survey section was adopted from a previous study [11]. A test/ re-test procedure was administered a week apart on pilot study subjects to calculate reliability of next questionnaire sections. Test/re-test correlation was 0.67 for physical activity section. Section 4 produced information on diet: for each of a list of 31 dietary and non-dietary items (including dairy products, baked products, meat, vegetables, fruits, and calcium supplements), the subjects were asked to estimate frequency of consumption (daily, weekly, fortnightly, or monthly). Similar checklists have been used in related previous studies [19]. Given the high coffee consumption in Central America, the subjects were asked about their degree of coffee consumption. Test/re-test correlation was 0.72 for dietary and non dietary calcium intake section. Final section (5) comprised eight open questions designed to assess nine knowledge dimensions about osteoporosis in subjects. The nine knowledge dimensions regarding osteoporosis, their specific questions and corresponding scores (from 0 to 42) are shown in Table 1. These questions served as a backbone for the interview and they were similar to other set used in a previous study [11]. Test/re-test correlation was 0.59 for osteoporosis knowledge questionnaire section. Data analysis Each subject's responses to the questions in the fifth section were analyzed by the principal researcher, who compared the answers with a semantic map (Fig. 1) developed by the authors on the basis of findings from previous studies on osteoporosis [1,2,5-7]. This approach allowed for quantification on knowledge about osteoporosis so: 5 points assigned for responses denoting knowledge of three or more concepts (each node in semantic map represents a concept), 3 points for responses denoting knowledge of 2 concepts, and 2 points for responses denoting knowledge about one out of osteoporosis-related concepts as reported previously [11]; except for osteoporosis information source dimension since its sub-score depended on number of information sources that women were able to mention, so that they only got one point per each source up to a maximum of 5 points. The total score achievable was 42 points. The total number of hours of weight-bearing physical activity was estimated by adding the subtotals for activities at home, activities at worksite, and that of weight-bearing exercises. Total calcium intake was estimated by adding the estimated subtotals for consumed dairy products, other dietary intake, and calcium supplements, including calcium content per portion × the number of portions per day. Obtained values were compared with reference values [20]. Data are cited in the text as means ± standard errors. For variables with homogeneous variance, means were compared by two-way analysis of variance and by Tukey tests for pairwise comparisons. For variables with non-homogenous variance, data were compared by the Kruskal-Wallis test (ANOVA on ranks) followed by Dunn's test for pairwise comparisons. These analyses were performed using SigmaStat version 2.03. Correlation analysis was used to determine relationships between knowledge scores and WBPA including exercise and calcium intake. To develop predictive models, we used a multivariate analysis based on multiple linear regression as contained in the program AMOS version 5 (Small Waters Corp.). Results Characteristics of the interviewed women The participant characteristics of each age group are shown in Table 2. Frequency of women with none or only a primary education increased with age; conversely, frequency of women with secondary or higher education decreased with age. Household income reported that most women (> 61%) were below the poverty line; although the number of women living on poverty conditions fluctuated with age. The frequency of nulliparity among interviewed women declined with age, whereas both the frequency of parity among interviewed women and the fecundity rate (children per woman) increased with age as anticipated. In all age groups, more than 44% of interviewed women reported a family history of osteoporosis, which included extended family: grandmothers, mothers, aunts, elder sisters, and cousins. Median, mean (SEM), and range of age of menopause in interviewed women were: 44, 42.3 (± 0.8), and 27–53 years, respectively. About 20% of interviewed women had lactose intolerance or ingested aluminum antiacids, both of these characteristics were unrelated of age. In addition, most women (> 69%) were coffee-consumers and the consumption of this drink increased with age. Knowledge about osteoporosis The sub-scores per knowledge dimensions about osteoporosis and age groups are shown in Table 3. The total scores of knowledge regarding osteoporosis were similar in the three age groups of women (median 14 in 25- to- 35-years; 17 in 36- to 49-years; 15 in over 49 years). Most of interviewed women (75%) had enough knowledge about osteoporosis regarding the concept of disorder and its risk factors, sex-related factor, and prevention behaviours, irrespective of age (Table 3). Conversely, these women got less knowledge scores for diagnosis and treatment of osteoporosis than other examined dimensions. In all three age groups, women with secondary or higher education obtained significantly higher total knowledge scores (F = 22.46, p < 0.001) than women with lower educational level (Fig. 2). There was not a significant relationship between age and educational level (F = 1.38, p = 0.223). Only 5% of the women with higher education obtained total scores of less than 12 points, and 75% of them obtained total scores of more than 25 (table 3). Multivariate analysis (factors age, educational level, family history of osteoporosis, household income, early menopause, fecundity) explained only about 37% of variance in total score (R2 = 0.367, p < 0.001) (Fig. 3). The most meaningful predictors of total score were educational level (R = 0.47, p < 0.001), household income (R = 0.18, p = 0.015), and early menopause (R = 0.18, p = 0.003). Physical activities The amount of weight-bearing physical activity was similar in the three age groups (mean 8.7 ± 0.5 hours/day in women 25- to 35 years; 8.9 ± 0.5 hours/day in those 36- to 49 years; 8.3 ± 0.6 hours/day in those over 49 years). Analysis of variance with factors of age and family history of osteoporosis indicated that neither factor produced a significant effect (F = 0.23, p = 0.794; F = 0.04, p = 0.852), nor the interaction between them was significant. The amount of weight-bearing physical activity reported at the worksite was markedly and significantly lower in over 49 years than in the other two age groups (H = 33.15, p < 0.001; Fig. 4). Conversely, physical activity at home was higher in women over-49 years than reported in the other two age groups (H = 19.34, p = 0.002; Fig. 5). The amount of weight-bearing exercise was low in all three groups, with means about 2 hours per week in the 25- to 35-years, and less than 1 hour per week in the older age groups (Fig. 6). About 75% of 25- to 35 years appear to do less than 2.7 hours of weight-bearing exercises per week; while about 75% of women in the older age groups reported doing no exercises (Fig. 6). However, the total duration of exercise did not vary significantly among the three age groups, or between women with or without a family history of osteoporosis (H = 10.25, p = 0.069). Multivariate analysis employing dependent variable of amount of total physical activity and candidate predictors age, educational level, household income, family history of osteoporosis, early menopause and fecundity, appeared to explain only 5% of total variance (R2 = 0.048, p = 0.038). Use of dependent variable of physical activity at worksite explained about 26% of variance (R2 = 0.264, p < 0.001; Fig. 7); the most effective predictors were educational level (R = 0.361, p < 0.01) and age (R = -0.234, p = 0.002). Similarly, use of dependent variable of physical activity at home explained about 27% of variance (R2 = 0.273, p < 0.001; Fig. 8); again the most effective predictor was age (R = -0.471, p < 0.001). Use of dependent variable of exercise activity explained only 13% of variance (R2 = 0.128, p < 0.001); the most effective predictor was family history of osteoporosis (R = 0.169, p = 0.011). Total osteoporosis knowledge score was not predicted on the amount of weight-bearing exercise (R = 0.081, p = 0.218). Although, total osteoporosis knowledge scores were significantly associated with WBPA at worksite (R2 = 0.07, p < 0.001), at home (R2 = 0.05, p = 0.002) or during exercise (R2 = 0.04, p = 0.004), squared correlations were rather low in all of cases. Dietary calcium intake Figure 9 summarizes the data on total calcium intake (mg/day) in the three age groups, subdivided into women above and below the poverty line. Independently of income, about 75% of women aged less than 49 years ingest less than 600 mg/day of calcium (i.e. only about 60% of the recommended daily intake, 1000 mg/day). In the over-49 age group (recommended daily intake 1200 mg/day), there is a marked difference in calcium intake between women above and below the poverty line; with women below the poverty line typically showing very low calcium intake (over 75% ingest less than 600 mg/day), whereas calcium intake in women above the poverty line is higher (though nevertheless lower than the recommended daily intake in over 75% of subjects). Calcium intake was estimated through the use of dairy products, with 75% of women ingesting less than 410 mg/day, in all three age groups below the poverty line, and in the two younger age groups above the poverty line. In the over-49 above-poverty-line group, 75% of women ingest less than 775 mg/day. Dairy products contributed on average 57% of total calcium intake in women below the poverty line, versus 68% of total calcium intake in women above the poverty line. Women in the over-49 above-poverty-line group ingested significantly more total calcium (H = 18.36, p = 0.003) and significantly more dairy calcium (H = 18.97, p = 0.002) than women in all other age/income groups (Figs. 10, 11). Supplementary calcium intake did not vary significantly among the age/income groups (H = 9.04, p = 0.108), neither was there any significant interaction between these factors. Multivariate analysis with dependent variable of total calcium intake and candidate predictors age, educational level, household income, lactose intolerance, coffee consumption and use of aluminum antiacids, provided a model that explained only 19% of total variance (R2 = 0.191, p < 0.001) (Fig. 12). With the addition of dairy calcium intake, the model explained 20% of total variance (R2 = 0.204, p < 0.001) (Fig. 13). In both cases, the most effective predictors were lactose intolerance (total calcium intake R = -0.239, p < 0.001; dairy calcium intake R = -0.272, p < 0.001), household income (R = 0.229, p = 0.003; R = 0.232, p = 0.002), age (R = 0.191, p = 0.005; R = 0.198, p = 0.004), and coffee consumption (R = -0.146, p = 0.025; R = -0.141, p = 0.029). Women who had moderate osteoporosis knowledge had an increase in their intake of calcium that was significant (R = 0.142, p = 0.045). Although total osteoporosis knowledge scores were significantly associated with total calcium intake (R2 = 0.06, p < 0.001) or dairy calcium intake (R2 = 0.04, p = 0.008) or non-dairy calcium intake (R2 = 0.03, p = 0.016) or calcium supplements (R2 = 0.07, p < 0.001), squared correlations were rather low in all cases. With the addition of calcium supplement intake only 12% of total variance was accounted for (R2 = 0.119, p = 0.003). The greatest predictors were a woman's use of aluminum antiacids (R = 0.232, p < 0.001) and her educational level (R = 0.207, p = 0.004). Discussion The total scores regarding nine knowledge osteoporosis dimensions were similar in all age groups of interviewed women (range of median 14 – 17, average 12.1 – 14.8) out of a possible 42 points. A similar study found Taiwanese women got a mean score of 15 out of 44 points related to six osteoporosis knowledge dimensions [21], whereas surveyed American women of three age groups got averaged knowledge scores from 32 to 44 points out of 183 [11]. In all cited cases, the obtained scores indicate that knowledge about osteoporosis is poor or limited among surveyed subjects so health educational programs and health services regarding osteoporosis are necessary for Salvadorean women, as it is also suggested for Taiwanese [21] and American women [11] of all ages. The present results also indicate that Salvadorean women with secondary or higher education have significantly better knowledge of osteoporosis than women with a low educational level, regardless of age. Similarly, other study found that better educated Chinese women in Singapore seem to know more about osteoporosis than those ones worst educated [22]. A previous study has likewise found that osteoporosis-related knowledge is independent of age [11]. Our multivariate analyses indicated that the most effective predictors of osteoporosis-related knowledge were educational level, household income, and early menopause. This latter factor perhaps affects osteoporosis-related knowledge through to give brochures and magazines to women at shopping centers, supermarkets, physician's clinics, schools, and colleges by non governmental organizations such as Salvadorean Demographical Association, dairy good producers such as New Zealand Dairy Board and Australian Milk Products, and some pharmaceutical laboratories, especially those produce calcium supplements. As well as information presented on television and in the press. Besides, short counselling sessions about preventive aspects of osteoporosis are given by some physician's private clinics, because there is not a settled public health education program about osteoporosis in El Salvador. This country is not only case in Latin America, since Mexican women got more osteoporosis information from mass communication media than health education activities of public institutions [14]. However, total knowledge about osteoporosis may not lead to an improvement in health lifestyle; it is necessary to know more about some specific aspects as osteoporosis risk factors and to acquire healthy habits to reduce the risk for low bone mass. In multivariate analyses, knowledge about osteoporosis was not a significant predictor of either amount of physical activity or total calcium intake. Previous studies likewise found that knowledge of osteoporosis does not correlate with risk-reducing life habits [11,12]. These authors suggested that osteoporosis-related knowledge among women of their sample was limited, the information was not fully understood or poorly internalized. They also suggested that it is unlikely that this type of knowledge will provide a basis for decisions about life style or unhealthy habits [11]. These comments are perhaps similarly applicable to the subjects in our study. Other previous studies have obtained results consistent with this view that osteoporosis-related knowledge is often poorly integrated and internalized, and does not lead to improved health behaviours [12,16,23,24]. Our data on physical activity in the three age groups indicate that the amount of physical activity engaged in paid work currently declines with increasing age, whereas the amount of physical activity during housework appears to increase with increasing age. This trend was confirmed by multivariate analysis, which indicates that the most effective predictors of amount of physical activity at worksite or at home were age and educational level. Educational level attained likely increases the likelihood of obtaining paid work. However, recreational activities and other health-preventive behaviours such as to do isometric exercises may also determine total physical activity. Considering all subjects together, most reported physical activity (96%) was performed in the course of daily housework, walking to work or shops, or standing at work or at home; only 4% was directly applicable to weight-bearing exercise. About 50% of women in the youngest age group did more than 2 hours of physical activity per week, but the amount of physical activity in the older women was markedly lower, and even in the youngest age group very few women performed the recommended daily minimum of 30 minutes of exercise per day [5,6]. Similar results were obtained in a previous study, which found that fewer than 50% of African-American and Hispanic women interviewed in the US do 60 minutes of physical exercise per week [16]. Very few of the women interviewed for the present study employ any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus perhaps reducing the risk of osteoporosis, such as isometrics [25]. Similar findings have been obtained in studies of Caucasian women in the US [11,12] and of African-American and Hispanic women residing in the US [12,16]. Independent of age and income, most women interviewed (50 – 75%) have a daily calcium intake of less than 60% of the recommended level, thus increasing the risk of osteoporosis. Similarly, in another studies of Caucasian and African-American and Hispanic women in the US [12,16], and Asian and Caucasian women in Australia [13], most of these women did not fulfil the suggested calcium intake. By contrast, in other recent study of Caucasian women in the US [11], only about 20% of women ingested less than 60% of the recommended daily calcium. The difference between our results in El Salvador and those obtained in the study of Caucasian women in the US [11] may, at least in part, reflect differences in standard of living between these two countries. Specifically, the generally higher incomes in the US may be associated with healthier diet, higher educational level, better access to healthcare, and better public education about general health and the prevention of diseases like osteoporosis. Additionally, calcium fortified foods are more readily available in the US. The average proportion of calcium intake as dairy products was 57% in women below the poverty line and 68% in women above the poverty line; both values are close to those reported for Caucasian women in the US [11]. These authors did not detect any significant variation with income, but note that their sample was smaller (n = 75) and more homogeneous (in terms of educational level and household income) than the sample of our study. The relationships between household income and calcium intake observed in our multivariate analyses likewise indicate that calcium intake varies with income. The other important predictor of calcium intake in this analysis was lactose intolerance, reported by 19% of the women interviewed; dairy calcium intake was likely lower in these women. As lightly lower prevalence of lactose intolerance (16%) was reported in the study of Caucasian women in the US [11]. The consumption of caffeine-containing drinks, especially coffee, has been shown to influence calcium intake, since these beverages often replace milk and milk-based beverages due to dairy products are relatively expensive in El Salvador and many Salvadorean families (42.9%) have a low purchasing power, and live below the poverty line [26]. Excessive caffeine ingestion is also reported to accelerate bone resorption [5,6], and prevents intestinal calcium absorption [9]. Calcium intake also varied with age, being significantly less among younger women. Similarly, most of interviewed Caucasian and African-American young adult women in the US did not get recommended calcium intake per day [12]. This may be because younger women tend to perceive dairy products as having a high content in animal fats, and thus tend to reject them, as it has been suggested by previous authors [19]. Auld et al. also suggested that Hispanic women (particularly adolescents and young women) in general tend to reject dairy products more than Anglosaxon and Asiatic women. The key practical implications of this study relate to suggest set up of primary healthcare programs regarding osteoporosis for Salvadorean women as it has also suggested for Taiwanese women [21], and Hispanic and African-American women [16], as well as training of healthcare professionals, which in light of the present study need to pay special attention to the following aspects of osteoporosis prevention: a) The type and frequency of physical exercise. b) Diet-related risk factors, including inadequate intake of calcium, vitamin D, and phosphorus; also the adverse effects of drinking coffee or other low-calcium drinks instead of milk or milk-based drinks. c) The importance of monitoring menstruation frequency, since normal circulating estrogen levels and normal menstrual cycles are important for maintaining normal bone metabolism. In addition, it is clearly important to set up health education programs to focus on integration and internalization of knowledge about osteoporosis. Although our results indicate that many women have modest knowledge about osteoporosis concerning risk factors and preventive behaviours, this knowledge often does not translate to appropriate changes in healthy life habits as it is shown through a weak association between total osteoporosis scores and exercise or calcium intake, so that osteoporosis knowledge is not well internalized among interviewed Salvadorean women. The lack of time to practice most adequate osteoporosis preventive exercise may accounts for the weak association between foregoing variables since majority of interviewed women perform WBPA (> 8 hours/day), but as labour activities or housework. The low calcium intake among subjects may be explained through they have a low purchasing power and do not have the habit to ingest enough dairy products, the main diet calcium source [9]. Non-dairy calcium intake including supplements was also low (< 370 mg/day), so that they did not fulfil the recommended daily calcium intake from this source either. Finally, we would suggest that osteoporosis education campaigns should be directed at families as well as individuals, with the aim of fomenting within-family education of children about the importance of calcium in the diet, whether as dairy products or in other calcium-rich products such soya milk and oat-based drinks and desserts. Conclusions It showed that Salvadorean women with secondary or higher education have significantly better knowledge about osteoporosis than women with lower educational level, regardless of age. However, this knowledge does not appear to lead to improved life style or preventive habits of osteoporosis. The amount of weight bearing physical activity (mainly walking or standing) during paid work or housework was high in the three age groups of interviewed women (over 8 hours/day). However, very few of the women interviewed for this study did not perform any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus reducing the risk of osteoporosis, such as going up and down stairs, jogging, aerobics, swimming, and isometrics. Most Salvadorean interviewed women have a daily calcium intake of 60% or less than the recommended level depending of lactose intolerance, household income, age, and coffee consumption. The former variables directly affect both calcium source diversity and intake frequency. Age would influence dairy calcium intake, since younger women tend to perceive dairy products as having a high animal fat content, and thus reject them. The coffee consumption would affect calcium intake, since this beverage often replace milk. Therefore, all these factors together may be increasing the risk to develop osteoporosis among Salvadorean women. List of abbreviations WBPA: Weight-bearing physical activity ANOVA: Analysis of Variance SEM: Standard error of mean Competing interests None declared. Authors' contributions RHR performed the design of the study, coordinated the study, performed data analysis, and drafted the paper. SMG participed in the design of the study and provided input into the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This research was supported by Universidad Andres Bello. The authors are indebted to Dr. Manuel Aldegunde and Ms. Veronica Sosa for help with the manuscript and they wish to thanks the health education worker, Mrs. Blanca Ruiz, and Nursing graduates: Mairena Escalante, Jacqueline Hernandez, Minerva Hernandez, Veronica Lozano, Alcira Portillo, Natalia Quijada, Damian Rodriguez, and Isabel Velasco for their skillful assistance with the survey administration. Figures and Tables Figure 1 Semantic map showing an osteoporosis knowledge structure. Semantic map showing an osteoporosis knowledge structure related to several aspect concerning to concept, risk-factors, prevention, and diagnosis of this skeletal disorder. Figure 2 Total osteoporosis knowledge scores. Total osteoporosis knowledge scores obtained by Salvadorean women in each age group (25 – 35 years, n = 73; 36 – 49 years, n = 74; over 49 years, n = 50), subclassified by educational level (none, primary, secondary, higher). Data are means ± SEM. Bars with distinct letters differ significantly within each age group (p < 0.05; two-way ANOVA followed by Tukey test). Figure 3 Predictive model of influences of surveyed variables on total knowledge scores. Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on total osteoporosis knowledge score (Tot_scor; R2 = 0.37, p < 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. Figure 4 Weight-bearing physical activities performed at worksite. Weight-bearing physical activity (hours/day) at worksite performed by Salvadorean women in each age group (25 – 35 years, n = 73; 36 – 49 years, n = 74; over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p < 0.05; Kruskal-Wallis' test followed by Dunn's test). Figure 5 Weight-bearing physical activities performed at home. Weight-bearing physical activity (hours/day) at home performed by Salvadorean women in each age group (25 – 35 years, n = 73; 36 – 49 years, n = 74; over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p < 0.05; Kruskal-Wallis' test followed by Dunn's test). Figure 6 Weight-bearing physical activity during exercise. Weight-bearing physical activity during exercise (hours/week) performed by Salvadorean women in each age group (Age 1: 25 – 35 years, n = 73; Age 2: 36 – 49 years, n = 74; Age 3: over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p < 0.05; Kruskal-Wallis' test followed by Dunn's test). Figure 7 Predictive model of influences of surveyed variables on WBPA performed at worksite. Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on amount of weight-bearing physical activities performed at worksite (WBPAwork; I; R2 = 0.26, p < 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. Figure 8 Predictive model of influences of surveyed variables on WBPA performed at home. Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on amount of weight-bearing physical activities performed at home (WBPAhome; II; R2 = 0.27, p < 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. Figure 9 Total calcium intakes among Salvadorean women classified by age and household income. Total calcium intakes among Salvadorean women in each age group (Age 1: 25 – 35 years, n = 73; Age 2: 36 – 49 years, n = 74; Age 3: over 49 years, n = 50), subclassified by household income (above or below the poverty line). Shaded boxes extend from the 25th to the 75th percentile; whiskers indicate the 5th and 95th percentiles; dots indicate individuals lying outside these percentiles; the mean is indicated by a solid line, the median by a dashed line. Figure 10 Total calcium intake among Salvadorean women classified by age and household income. Total calcium intake among Salvadorean women in each age group (25 – 35 years, n = 73; 36 – 49 years, n = 74; over 49 years, n = 50), subclassified by household income (above or below the poverty line). Data are means ± SEM. Bars with distinct letters differ significantly (p < 0.05; Kruskal-Wallis' test followed by Dunn's test). Figure 11 Dairy calcium intake among Salvadorean women classified by age and household income. Dairy calcium intake among Salvadorean women in each age group (25 – 35 years, n = 73; 36 – 49 years, n = 74; over 49 years, n = 50), subclassified by household income (above or below the poverty line). Data are means ± SEM. Bars with distinct letters differ significantly (p < 0.05; Kruskal-Wallis' test followed by Dunn's test). Figure 12 Predictive model of influences of surveyed variables on total calcium intake. Predictive model of the surveyed variables: age (Age), lactose intolerance (Lactose), coffee consumption (Coffee), use of aluminum antiacids (Antiacid), household income (Income), and education (Educatn) influencing on total calcium intake (Total_Ca; I; R2 = 0.19, p < 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. Figure 13 Predictive model of influences of surveyed variables on dairy calcium intake. Predictive model of the surveyed variables: age (Age), lactose intolerance (Lactose), coffee consumption (Coffee), use of aluminum antiacids (Antiacid), household income (Income), and education (Educatn) influencing on dairy calcium intake (Dairy_Ca; II; R2 = 0.20, p < 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. Table 1 Knowledge dimensions about osteoporosis, specific questions, and corresponding scores get through concept integration. Knowledge dimensions about osteoporosis and corresponding questions None related concept, sub-scores One related concept, sub-scores Up to two related concepts, sub-scores ≥ Three related concepts, sub-scores Concept related What you can tell me about osteoporosis? 0 2 3 5 Symptom related What changes do you note in your body if you have osteoporosis? 0 2 3 5 Affected bones If one bone or more was mentioned when previous question was done. 0 2 3 5 Sex-related factor Are women and men equally prone to osteoporosis, or is it more frequent in one of the sexes? 0 2 --- --- Risk-factor related What factors increase the risk of suffering osteoporosis? 0 2 3 5 Prevention behavior related How can you reduce the risk of suffering osteoporosis? 0 2 3 5 Diagnosis related How can osteoporosis be detected? 0 2 3 5 Actual treatment related How can osteoporosis be treated? 0 2 3 5 Osteoporosis information source Where did you get the information about osteoporosis? 0 2 3 5 Maximum score (Σ) 42 Table 2 Descriptive data from a convenience sample, according to women's age-group. Characteristics 25–35 years (n = 73) 36–49 years (n = 74) over 49 years (n = 50) Age-Mean (SEM) 29.8 (0.4) 42.4 (0.5) 64.5 (1.5) Educational level (%) None 3 (4.1) 15 (20.3) 14 (28.0) Primary 23 (31.5) 27 (36.5) 25 (50.0) Secondary 17 (23.3) 9 (12.1) 4 (8.0) Higher 30 (41.1) 23 (31.1) 7 (14.0) Household income (%) Above the poverty line 28 (38.4) 28 (37.8) 13 (26.0) Below the poverty line 45 (61.6) 46 (62.2) 37 (74.0) Fecundity (%) Children per woman†-Mean (SEM) 2.4 (0.2) 2.7 (0.1) 6.0 (0.5) Nulliparity 16 (21.9) 5 (6.8) 5 (10.0) Parity 57 (78.1) 69 (93.2) 45 (90.0) Family history of osteoporosis (%) Yes 40 (54.8) 33 (44.6) 32 (64.0) No 33 (45.2) 41 (55.4) 18 (36.0) Menopause among women (Irrespective of age group). Mean age (years) 42.3 ± 0.8 Median age (years) 44.0 Range of age (years) 27.0 – 53.0 Menopause before age 45 years (%) 25–35 years (n = 73) 36–49 years (n = 74) Over 49 years (n = 50) Yes 4 (5.5) 18 (24.3) 18 (36.0) No 69 (94.5) 56 (75.7) 30 (64.0) Use of aluminum antiacids (%) Users 16 (21.9) 17 (23.0) 13 (26.0) Non-users 57 (78.1) 57 (77.0) 37 (74.0) Lactose intolerance (%) Yes 15 (20.5) 14 (18.9) 9 (18.0) No 58 (79.5) 60 (81.1) 41 (82.0) Coffee consumption (%) Yes 51 (69.9) 62 (83.8) 39 (78.0) No 22 (30.1) 12 (16.2) 11 (22.0) Cups a day‡-Mean (SEM) 1.8 (0.1) 2.1 (0.1) 1.9 (0.1) † Fecundity rate is only based on data of parity per each age group. ‡ Consumption rate is only based on coffee-consumer data per each age group. Table 3 Osteoporosis knowledge scores according knowledge dimensions and women's age-groups. Knowledge dimensions about osteoporosis (expressed as scores).† 25–35 years (n = 73)a 36–49 years (n = 74)a Over 49 years (n = 50)a Concept related 3 (5) 3 (5) 3 (5) Symptom related 2 (3) 3 (3) 3 (3) Affected bone related 0 (3) 2 (2) 0 (2) Sex related 2 (2) 2 (2) 2 (2) Risk factor related 3 (5) 3 (5) 3 (5) Prevention behavior related 3 (5) 3 (5) 3 (5) Diagnosis related 0 (3) 0 (3) 0 (3) Actual treatment related 0 (0) 0 (0) 0 (0) Osteoporosis information source 1 (2) 1 (2) 1 (2) Σ 14 (28) 17 (27) 15 (27) † All values are median and 75th percentile (in brackets) of reached scores in each knowledge areas, per each age group. a Comparisons among scores of knowledge specific area per each age group were not significantly different (p > 0.05, Kruskal-Wallis ANOVA on ranks). ==== Refs Ramos F Ramos F Osteoporosis In El Paciente Reumático 2001 3,4 México: Ciencia y Cultura Latinoamericana 67 114 Rosenberg A Cotran RS, Kumar V, Collins T Osteoporosis In Robbins Patología Estructural y Funcional 2000 6 Madrid: McGraw-Hill Interamericana 1266 1268 Campusano C Cerdas S Latin American Osteoporosis Advisory Board Metabolismo óseo y fisiopatología de la osteoporosis In Curso Latinoamericano sobre Osteoporosis 2003 5 México: Programa Latinoamericano de Educación Médica Continuada-Aventis 16 23 Galué G de la Peña P Latin American Osteoporosis Advisory Board Diagnóstico de la osteoporosis: aspecto clínico, laboratorial y de imagen In Curso Latinoamericano sobre Osteoporosis 2003 México: Programa Latinoamericano de Educación Médica Continuada-Aventis 5 13 ¿Qué es la Osteoporosis? La Osteoporosis Calcium and Osteoporosis National Institutes of Health Osteoporosis prevention, diagnosis, and therapy NIH Consensus Statements 2000 17 1 45 Jasqui S Man Z Latin American Osteoporosis Advisory Board Tratamiento y prevención de la osteoporosis: terapéutica estro-progestativa In Curso Latinoamericano sobre Osteoporosis 2003 México: Programa Latinoamericano de Educación Médica Continuada-Aventis 5 11 Suleiman S Nelson M Li F Buxton M Moniz C Effect of calcium intake and physical activity level on bone mass and turnover in healthy, white, postmenopausal women Am J Clin Nutr 1997 66 937 943 9322571 Terrio K Auld G Osteoporosis knowledge, calcium intake, and weight-bearing physical activity in three age groups of women J Community Health 2002 27 307 320 12238730 10.1023/A:1019840709367 Kasper MJ Peterson MG Allegrante JP The need for comprehensive educational osteoporosis prevention programs for young women: results from a second osteoporosis prevention survey Arthritis Rheum 2001 45 28 34 11308058 10.1002/1529-0131(200102)45:1<28::AID-ANR80>3.3.CO;2-7 Liew YL Mann D Piterman L Osteoporosis risks. A comparative study of Asian Australian and Caucasian Australian women [abstract] Aust Fam Physician 2002 31 291 293 11926162 Velasco-Murillo V Navarrete-Hernández E Ojeda-Mijares RI Pozos-Cavanzo JL Camacho-Rodríguez MA Cardona-Pérez JA Experiencia y conocimiento sobre climaterio y menopausia en mujeres de la ciudad de México Gac Med Mex 2000 136 555 564 11131857 Orces CH Casas C Lee S Garci-Cavazos R White W Determinants of osteoporosis prevention in low-income Mexican-American women South Med J 2003 96 458 464 12911184 10.1097/01.SMJ.0000051905.38128.B4 Geller SE Derman R Knowledge, beliefs, and risk factors for osteoporosis among African-American and Hispanic women J Natl Med Assoc 2001 93 13 21 12653376 Diez Primeras Causas más Frecuentes de Morbilidad, Atendidas en Consulta Ambulatoria Indicadores Económicos Auld G Boushey CJ Bock MA Bruhn C Gabel K Gustafson D Holmes B Misner S Novotny R Peck L Pelican S Pond-Smith D Read M Perspectives on intake of calcium-rich foods among Asian, Hispanic, and white preadolescent and adolescent females J Nutr Educ Behav 2002 34 242 251 12559059 Dietary Reference Intakes: recommended levels for individual intake Yu S Huang YC Knowledge of, attitudes toward, and activity to prevent osteoporosis among middle-aged and elderly women J Nurs Res 2003 11 65 72 12695981 Saw SM Hong CY Lee J Wong ML Chan MF Cheng A Leong KH Awareness and health beliefs of women towards osteoporosis Osteoporos Int 2003 14 595 601 12830368 10.1007/s00198-003-1403-6 Ribeiro V Blakeley J Laryea M Women's knowledge and practices regarding the prevention and treatment of osteoporosis Health Care Women Int 2000 21 347 353 11813780 10.1080/073993300245195 Ailinger R Emerson J Women's knowledge of osteoporosis Appl Nurs Res 1998 11 111 114 9757610 Swezey RL Swezey A Adams J Isometric progressive resistive exercise for osteoporosis J Rheumatol 2000 27 1260 1264 10813298 Indicadores de Pobreza y Hogares Múltiples

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BMC Musculoskelet Disord. 2004 Aug 26; 5:29

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BMC Musculoskelet Disord

10.1186/1471-2474-5-29

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==== Front BMC SurgBMC Surgery1471-2482BioMed Central London 1471-2482-4-91534742710.1186/1471-2482-4-9Case ReportGraft calcifications and dysfunction following liver transplantation Tzimas George N [email protected] Mohammad [email protected] Eric [email protected] Anouk [email protected] Hojatollah [email protected] Peter P [email protected] Division of General Surgery/ Section of Hepatobiliary and Transplantation Surgery, Royal Victoria Hospital, McGill University Health Center, 687 Pine Avenue West, S 10.26, Montreal, Quebec, H3A 1A1, Canada2 Facility for Electron Microscopy Research, Strathcona Medical Building, 3640 University Street, McGill University, Montreal, Quebec, H3A 2B2, Canada3 Organelle Signaling Laboratory, Department of Surgery, Royal Victoria Hospital, H 6.34, McGill University, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1, Canada2004 3 9 2004 4 9 9 26 11 2003 3 9 2004 Copyright © 2004 Tzimas et al; licensee BioMed Central Ltd.2004Tzimas et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The molecular events, following ischemia and reperfusion (I/R) of the liver during transplantation are largely unknown. There is evidence that apoptotic and necrotic events may take place, and occasionally result in primary graft dysfunction. We herein report two cases, where significant I/R injury correlated with the development of liver calcification and primary liver dysfunction. Case Presentation Both patients with clinical and biochemical evidence of primary graft dysfunction demonstrated calcification at light and electron microscopy levels. In addition, one patient had macroscopic evidence of calcification on cross-sectional imaging. Both patients died secondary to the sequelae of the graft dysfunction. Conclusions Severe I/R-induced injury to the liver, clinically leads to graft dysfunction. This is due to advanced apoptotic and/or necrotic events at the hepatocyte level that may, on the most severe form, lead to calcification. The study of microcalcification at the early posttransplant period could provide insight in the events taking place following significant ischemia/reperfusion-induced injury to the graft. Liver TransplantationIschemiaReperfusionGraft DysfunctionOutcome ==== Body Background Liver transplantation (LT) remains the only treatment for end-stage liver disease. However the transplant procedure mandates cold perfusion, hypothermic storage, warm ischemia and warm reperfusion of the graft, resulting in Ischemia/Reperfusion (I/R)-induced injury to the transplanted graft. Although the introduction of the University of Wisconsin solution (UW) has improved clinical outcomes, I/R injury remains one of the major clinical problems following LT leading, in cases of marginal donor quality or prolonged cold or warm ischemic time, to the development of graft dysfunction or non function. Recently the cellular events following liver I/R during LT have been brought to sharp focus. Nevertheless, it is still controversial if the major mode of cell death during I/R is apoptosis and/or necrosis [1]. It has been shown in an animal model of viral-induced hemorrhagic liver necrosis, that the liver undergoes several morphologic changes including mineralization [2]. Furthermore, previous clinical reports have documented the development of calcifications in liver upon extensive ischemia [3,4]. The above observations may reflect a high degree of cell damage leading to an enhanced apoptotic cell engulfment by non-professional phagocytes of the liver [5] with subsequent necrosis and biomineralization. In this report, using a combination of cross-sectional imaging, histochemistry, and light (LM) as well as transmission electron microscopy (TEM) we identified liver calcification, in two grafts with severe I/R injury following LT. Case Presentations Case 1 A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution's transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis. The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP). At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L (Figure 1A,1B). On postoperative day 2 the patient had a peak of his serum AST (3469 U/L) and at this point he had further biochemical evidence of primary graft dysfunction, with inability to normalize his INR (Figure 1B), and with progressive elevation of his total serum bilirubin (Figure 1A). Repeated ultrasonographic examination revealed a patent hepatic artery and portal vein, as well as patent hepatic veins. At this point, a liver biopsy demonstrated severe reperfusion injury with several apoptotic bodies, several dystrophic calcifications (Figure 1E), and no evidence of acute cellular rejection. His clinical status deteriorated, he developed multiorgan system failure and died 12 days after his transplantation. No septic focus was identified. Both kidneys harvested from the same donor did not present any signs of delayed graft function after transplantation. Figure 1 A: Serum Bilirubin levels (μmol/L) for patient 1 (solid line) and for patient 2 (dashed line). The arrowheads indicate the time points where calcifications were detected. B: International Normalization Ratio (INR) for patient 1 (solid line) and for patient 2 (dashed line). Arrowheads indicate the time points where calcifications were detected. C: Computed Tomography of patient 2. The arrow points an area in the right liver with the same density as the spinal column (arrowhead). D: Picture of the explant liver during retransplantation of the same patient (case 2). The arrow shows the abnormal area of the right liver correlating with the computed tomography findings. E: Light microscopy of epoxy embedded semi-section obtained from the liver biopsy from patient 1. The image shows moderate calcification (microcalcification) throughout the section. The arrow indicates a representative pattern of calcification. F: Light microscopy of epoxy embedded semi-section obtained from the tissues of the explant, following liver retransplantation of patient 2. The image shows the interface between calcified region (upper right) and non-calcified adjacent hepatic cells (lower left region). G: Light microscopy image showing a higher magnification of calcified region as shown in F. The bright and high contrast regions represent massive mineralization of hepatic cells of the explant, following retransplantation. H: Transmission electron microscopy images of ultrathin section obtained from the transitional zone between calcified and non-calcified tissue. Showing the mode of calcification and textural organization of hydroxyapatite crystal aggregates (dark contrast) within cytoplasmic region of the cell. Note alteration of the nucleus in the center. Case 2 A 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP< 90 mm Hg). At the time of LT, the patient had an INR of 2.7 and a serum total bilirubin of 104 μmol/L. Following LT, the patient developed severe primary graft dysfunction with rising serum bilirubin and INR (Figure 1A,1B), while his serum ALT and AST peaked during the 2nd and 3rd postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels (hepatic artery, portal and hepatic veins), while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column (Figure 1C). The patient was listed for retransplantation. During the retransplant procedure the explant liver graft had a "bony" consistency in the involved right lobe. Cross-sections of the right lobe showed a "clay-like" parenchyma with clear evidence of calcification (Figure 1D). Light (LM) and transmission electron microscopy (TEM) investigation of ultrathin sections obtained from specimens selected from biopsies at the interface of calcified and non-calcified tissues showed extensive intracellular calcification within the hepatic cells (Figure 1F). High-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) combined with energy dispersive spectroscopy (EDS) analysis demonstrated the presence of hydroxyapatite (HA) as a solid phase in the calcified region. The adjacent non- or partially calcified hepatic cells displayed extensive nuclear condensation suggestive of significant apoptosis as well as severe vacuolization, suggestive of an extensive apoptotic and necrotic process (Figure 1G,1H). The patient had a complicated postoperative course and finally died from ventricular fibrillation unresponsive to electrical cardioversion. Both kidneys harvested from the initial donor were transplanted without any evidence of delayed or primary graft dysfunction/non-function. Conclusions Currently, more than 16,000 candidates are listed with the United Network for Organ Sharing awaiting liver transplantation. Nevertheless, only 4800 cadaveric liver transplants are performed annually in the United States. Due to this discordance between organ demand and supply, it is estimated that approximately 10% of patients in the waiting list will die before obtaining an organ. As a result, novel strategies to expand the donor pool have been explored. With the exception of live donor liver transplantation, the remaining strategies involve the use of older cadaveric grafts, allografts with mild steatosis or even donors with evidence of past hepatitis B or C infection [6]. This is why, one of the major obstacles to be tackled, is the development of clinically significant ischemia and reperfusion injury, which is even more important for "marginal" grafts. Every progress towards understanding the molecular events following not only cold storage but also cold and warm reperfusion of the graft could have a significant impact on the current transplantation practices. Indeed, recent data suggest that following I/R there is a balanced apoptosis and occasionally necrosis of hepatocytes translating into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy [7]. Furthermore, when these events become predominant, they can lead, at least in animal models, to the development of calcifications as observed in livers of rabbits infected with rabbit haemorrhagic virus [2]. In the present case report, we have shown that these events can take place in human subjects following liver transplantation. To our knowledge these are the first reported cases in the literature of liver calcification following liver transplantation, presumably secondary to I/R injury. Not only did both patients have biochemical evidence of severe graft I/R injury, they also had biopsy proven I/R induced injury associated with the development of calcifications. Furthermore, both succumbed to the sequelae of this injury. Both recipients received grafts from donors with normal serum biochemistries and no evidence of hepatic trauma or steatosis. Both donors had no evidence of crystal deposition or storage disease, and although we did not perform any donor liver biopsies, the grafts appeared macroscopically normal and perfused well with UW solution. Corroborating to this remains the fact that all four kidneys (from both donors) were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination. Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification [8]. Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage [9], as well as synthetic HA formed in serum [10], the intracellular precipitation of HA within hepatic cells is unique and has not been reported from other physiological and pathological tissues. The observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver. In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant. Competing Interests None declared. Authors Contributions GNT: conceived the study and wrote the manuscript MA: carried out the electron microscopy studies EC: participated in the design and analyzed the light microscopy results, critical review of the manuscript AE: analyzed light microscopy results AV: carried out electron microscopy studies PM: participated in the design of the study All authors read and approved the final manuscript. Abbreviations I/R: Ischemia and Reperfusion LM: Light Microscopy TEM: Transmission Electron Microscopy HRTEM: High-resolution Transmission Electron Microscopy EDS: Energy Dispersive Spectroscopy HA: Hydroxyapatite Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Mrs. J. Mui (Senior Technician, Facility for Electron Microscopy Research, McGill University) for her technical assistance. GNT was supported by the Gertrude Nemiroff Eliesen Memorial Award, and EC by the Simone and Morris Fast foundation, McGill University Health Center Research Institute and a Junior scholarship from the FRSQ. This work was supported in part by a grant from the Canadian Institutes for Health Research (CIHR) to EC and National Science and Engineering Research Council of Canada (NSERC) to HV. Consent was obtained from patient relative for publication. ==== Refs Meguro M Katsuramaki T Kimura H Isobe M Nagayama M Kukita K Nui A Hirata K Apoptosis and necrosis after warm ischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714 Transplantation 2003 75 703 710 12640313 10.1097/01.TP.0000053400.42842.5C Teifke JP Reimann I Schirrmeier H Subacute liver necrosis after experimental infection with rabbit haemorrhagic disease virus (RHDV) J Comp Pathol 2002 126 231 234 11945014 10.1053/jcpa.2001.0534 Shibuya A Unuma T Sugimoto T Yamakado M Tagawa H Tagawa K Tanaka S Takanashi R Diffuse hepatic calcification as a sequela to shock liver Gastroenterology 1985 89 196 201 4007404 Shih WJ Han JK Magoun S Wierzbinski B Bone agent localization in hepatic metastases J Nucl Med Technol 1999 27 38 40 10322573 Dini L Pagliara P Carla EC Phagocytosis of apoptotic cells by liver: a morphological study Microsc Res Tech 2002 57 530 540 12112436 10.1002/jemt.10107 Velidedeoglu E Desai NM Campos L Olthoff KM Shaked A Nunes F Zeldin G Stewart C Blumberg E Abrams J Markmann JF The outcome of liver grafts procured from hepatitis C-positive donors Transplantation 2002 73 582 587 11889435 10.1097/00007890-200202270-00018 Syntichaki P Tavernarakis N Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos? EMBO Rep 2002 3 604 609 12101090 10.1093/embo-reports/kvf138 Trump BF Berezesky IK Laiho KU Osornio AR Mergner WJ Smith MW The role of calcium in cell injury. A review Scan Electron Microsc 1980 437 62, 492 6999604 Hall Brian Keith Newman Stuart Cartilage : molecular aspects 1991 Boca Raton, Fla., CRC Press 268

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BMC Surg. 2004 Sep 3; 4:9

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BMC Surg

10.1186/1471-2482-4-9

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==== Front BMC BiotechnolBMC Biotechnology1472-6750BioMed Central London 1472-6750-4-181533101610.1186/1472-6750-4-18Research ArticleHeterologous expression of plant virus genes that suppress post-transcriptional gene silencing results in suppression of RNA interference in Drosophila cells Reavy Brian [email protected] Sheila [email protected] Tomas [email protected] Stuart A [email protected] Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2004 25 8 2004 4 18 18 22 5 2003 25 8 2004 Copyright © 2004 Reavy et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background RNA interference (RNAi) in animals and post-transcriptional gene silencing (PTGS) in plants are related phenomena whose functions include the developmental regulation of gene expression and protection from transposable elements and viruses. Plant viruses respond by expressing suppressor proteins that interfere with the PTGS system. Results Here we demonstrate that both transient and constitutive expression of the Tobacco etch virus HC-Pro silencing suppressor protein, which inhibits the maintenance of PTGS in plants, prevents dsRNA-induced RNAi of a lacZ gene in cultured Drosophila cells. Northern blot analysis of the RNA present in Drosophila cells showed that HC-Pro prevented degradation of lacZ RNA during RNAi but that there was accumulation of the short (23nt) RNA species associated with RNAi. A mutant HC-Pro that does not suppress PTGS in plants also does not affect RNAi in Drosophila. Similarly, the Cucumber mosaic virus 2b protein, which inhibits the systemic spread of PTGS in plants, does not suppress RNAi in Drosophila cells. In addition, we have used the Drosophila system to demonstrate that the 16K cysteine-rich protein of Tobacco rattle virus, which previously had no known function, is a silencing suppressor protein. Conclusion These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi. ==== Body Background RNA interference (RNAi) is a process in which the introduction of dsRNAs into cells leads to inactivation of expression of genes containing homologous sequences by sequence-specific degradation of mRNA (for reviews see [1,2]). RNAi has been observed in a variety of organisms, including fruit fly, nematodes, zebrafish, mice and humans [3-7], and is mechanistically similar to post-transcriptional gene silencing (PTGS) in plants [8] and quelling in fungi [9]. Genetic studies have identified a number of the proteins that are involved in these processes in Neurospora crassa [10-12], Caenorhabditis elegans [13-15] and Arabidopsis thaliana [16-18]. dsRNA has been shown to induce RNAi in cultured Drosophila cells [19-22] and biochemical studies of this system have revealed the involvement of two distinct activities; an RNA-induced silencing complex (RISC), containing both nuclease activity and RNA that carries out enzymatic degradation of target RNA [19], and an RNase III-like protein (Dicer) that is involved in production of short (22 nucleotide) guide RNAs from dsRNA as an early step in the RNAi process [22]. PTGS in plants can operate as a defence mechanism against virus infection (for reviews see [8,23]) and numerous plant viruses encode silencing suppressors, which are thought to have developed as a response to the plant PTGS system [24]. Plant virus-encoded silencing suppressors may target different components of the PTGS system. The HC-Pro protein, encoded by potyviruses such as Tobacco etch virus (TEV), was initially characterised as a determinant of virus movement in plants [25] and subsequently was shown to inhibit silencing of transgenes in transformed plants [26]. HC-Pro antagonizes silencing in all tissues [26] and appears to target a step involved in the maintenance of silencing [27]. In contrast, the 2b protein encoded by Cucumber mosaic virus (CMV), a cucumovirus, interferes with the systemic spread of the silencing signal, preventing initiation of silencing in newly emerging tissues [28]. A survey of a small number of other different plant viruses showed that the comovirus Cowpea mosaic virus, the geminivirus African cassava mosaic virus, the potexvirus Narcissus mosaic virus, the tobamovirus TMV, the sobemovirus Rice yellow mottle virus (RYMV), the tombusvirus Tomato bushy stunt virus (TBSV) and the tobravirus Tobacco rattle virus (TRV) also were able to suppress GFP silencing [24]. Although in this study the potexvirus PVX did not suppress silencing, using a different assay these authors showed that PVX is in fact able to prevent systemic silencing [29]. Thus, it seems probable that many plant viruses encode proteins that allow them to evade or inhibit PTGS in certain plant species, and that different suppressor proteins target different parts of the PTGS pathway. An amenable system for studying these suppressor proteins would be an aid in determining the molecular basis of their action. Recently it has been demonstrated that an insect virus, flock house virus (FHV) encodes a protein, 2b, which acts as a suppressor of PTGS in plants [30]. We have investigated the effects on RNAi in cultured Drosophila cells of expression of some silencing suppressors from plant viruses. Results Effects of transient expression of the TEV HC-Pro and CMV 2b proteins on RNAi in Drosophila cells The TEV HC-Pro or CMV 2b proteins were transiently expressed in Drosophila DS2 cells and their effect on dsRNA-mediated silencing of a lacZ gene examined using a previously described system [20]. Expression of the CMV 2b protein could be detected by immunoblotting in lysates of cells transfected with pMT-2b and induced by addition of CuSO4 to the growth medium (Fig 1A, lane 4). No cross-reacting protein was detected in similarly induced cells not transfected with pMT-2b (Fig 1A, lane 2) or in control cells, or cells transfected with pMT-2b that were not induced (Fig 1A, lanes 1 and 3. In the absence of an antibody to TEV HC-Pro we used an expression plasmid (pMT-HC-Pro/K) that expressed a mutated HC-Pro (TEV K) containing an insertion of 3 amino acids in the central region of the protein [31] as a negative control to verify that any suppressor activity was due to an effect of the HC-Pro protein. This mutant version of HC-Pro was shown previously to be defective in suppression of PTGS in plants [32]. Northern blot analysis of RNA from cells transfected with pMT-HC-Pro or pMT-HC-Pro/K showed that transcript from both plasmids accumulated in the cells (Figure 1B, lanes 2 and 3). β-galactosidase activity could be detected by staining in ~80% of Drosophila cells when they were transfected with a lacZ expression plasmid (pMT/V5-His/lacZ) (Table 1). ~17% of cells stained for β-galactosidase when they were co-transfected with pMT/V5-His/lacZ and dsRNA corresponding to the first ~500nt of the lacZ gene demonstrating induction of RNAi. No significant decrease in the number of cells stained was observed when cells were transfected with pMT/V5-His/lacZ and dsRNA derived from the green fluorescent protein gene indicating that the silencing was specific. Co-transfection of Drosophila cells with pMT/V5-His/lacZ, lacZ-specific dsRNA and an HC-Pro expression vector (pMT-HC-Pro) resulted in staining of ~46% of cells, indicating that RNAi was being suppressed. Transfection of cells with the mutant pMT-HC-Pro-K along with pMT/V5-His/lacZ and dsRNA resulted in no suppression of RNAi. This indicates that a mutation affecting the ability of HC-Pro to suppress PTGS in plants also affects suppression of RNAi in Drosophila cells. Transfection of cells with pMT-2b along with pMT/V5-His/lacZ and dsRNA resulted in no increase in the percentage of transfected cells staining for β-galactosidase activity compared to cells transfected only with pMT/V5-His/lacZ and dsRNA. RNAi in stable cell lines expressing TEV HC-Pro or CMV 2b Stable cell lines expressing HC-Pro (DS2-HC-Pro), or CMV 2b (DS2-2b) were produced in order to improve the efficiency of the RNAi suppression assay by reducing the number of different nucleic acid molecules needed for co-transfection. Immunoblotting of lysates of DS2-2b cells confirmed that the 2b protein accumulated in these cells (Fig 2A, lane 8) and this protein was not produced when the cells were not treated with CuSO4 (Fig 1A, lane 7). Northern blot analysis of RNA from the DS2-HC-Pro cells showed that an HC-Pro-specific transcript accumulated in these cells (Fig 1C, lane 1). An unrelated cell line (DS2-scAb S20) expressing a recombinant antibody [33] (Reavy et al., 2000) was used as a control in order to eliminate the possibility that stable transformation of the cells could interfere with the RNAi mechanism. The transfection efficiency of all transformed lines with pMT/V5-His/lacZ was lower than that of the control DS2 cells (Table 1); possibly the transformed cells are more recalcitrant to transfection than the control cells or the additional copies of the metallothionein promoter are saturating the induction factors. Nonetheless, RNAi was strongly induced in the DS2-scAb S20 cells as co-transfection with pMT/V5-His/lacZ and dsRNA reduced the number of cells staining for β-galactosidase activity to only 6.5% compared to ~41% when transfected with pMT/V5-His/lacZ alone (Table 1). Transfection of DS2-HC-Pro cells with pMT/V5-His/lacZ resulted in ~42% of cells expressing β-galactosidase. This was reduced only to ~32% when transfection was carried out using pMT/V5-His/lacZ and dsRNA, showing that RNAi was significantly inhibited in this cell line. In contrast, no suppression of RNAi was observed in the DS2-2b line (expressing the CMV 2b protein) when transfected with pMT/V5-His/lacZ and dsRNA, as the percentage of stained cells was similar to that in DS2-scAb S20 cells transfected with pMT/V5-His/lacZ and dsRNA. Similar results were obtained when the DS2-2b cells were grown in the presence of CuSO4 to induce expression of the virus protein before transfection with pMT/V5-His/lacZ and dsRNA, indicating that the CMV 2b protein could suppress neither the initiation nor the maintenance of the dsRNA-induced silencing in the Drosophila cells. Identification of silencing suppression in Drosophila cells by another plant virus protein We were interested to determine if the Drosophila cell system could be used as a screen for RNAi suppression effects caused by other virus proteins. We chose to examine the potential RNAi suppression activity of a protein from the tobravirus TRV. This virus is able to suppress transgene silencing in plants [24] but the specific viral protein responsible for this activity has not been identified. TRV, like the other tobraviruses has a bipartite, positive strand RNA genome [34], however, the larger RNA (RNA1) can infect plants systemically in the absence of RNA2 to produce what is known as an NM-infection. This occurs frequently in particular cultivars of potato and is often associated with increased symptom severity. Clearly, therefore, RNA1 encodes all the functions necessary for virus multiplication including, possibly, suppression of PTGS/host defence. The one protein encoded by RNA1 without an assigned function is a 16K cysteine-rich protein which was, thus, investigated as a candidate silencing suppressor protein. One characteristic of plant virus silencing suppressor proteins is often significant enhancement of disease symptoms when they are over-expressed from a viral vector [26]. Similar results were obtained from preliminary experiments showing that expression of the TRV 16K gene from a PVX vector did increase the severity of symptoms in infected plants. Inoculation of Nicotiana benthamiana plants with PVX alone initially induced vein chlorosis and systemic leaf curling, although the plants continued to grow. Inoculation with PVX carrying the 16K gene caused similar initial symptoms but led to tip necrosis and death of the plants [35]. The TRV 16K protein was expressed in Drosophila cells after transfection of cells with the expression plasmid pMT-16K, and the 16K protein could be detected by western blotting when the cells were induced with CuSO4 (Fig 2, lane 4) but not when cells were not induced (Fig 2, lane 3). A stably-transformed cell line (DS2-16k) containing pMT-16k behaved in a similar way (Fig 2, lanes 7, 8). No cross-reacting protein was detected in non-transfected cells (Fig 2, lanes 1,2). Co-transfection of pMT/V5-His/lacZ with dsRNA and pMT-16K resulted in ~47% of transfected cells staining blue (Table 1) compared to ~18% when transfected with pMT/V5-His/lacZ and dsRNA. The 16K protein therefore partially suppressed RNAi in Drosophila cells. Detection of lacZ gene transcripts Northern blot analysis of the RNA present in transfected cells confirmed that HC-Pro was effective in preventing cytoplasmic degradation of the lacZ transcript. RNA with the expected size of the lacZ transcript could not be detected in extracts of DS2 cells that were transfected with pMT/V5-His/lacZ and dsRNA (Fig. 3, lane 2). However, intact lacZ RNA was present in extracts of DS2-HC-Pro cells that were transfected with pMT/V5-His/lacZ regardless of whether the cells were co-transfected with dsRNA (Fig 3, lanes 3 & 4). Similarly, the lacZ transcript was intact in DS2-16K cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig 3, lane 6). The amounts of lacZ transcript in the DS2-Hc-Pro and DS2-16K cells were less after transfection with pMT/V5-His/lacZ and dsRNA than after transfection with pMT/V5-His/lacZ alone indicating partial suppression of RNAi. No lacZ transcript was detected in DS2-2b cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig 3, lane 10) but the lacZ transcript could be detected in DS2-2b cells transfected with pMT/V5-His/lacZ alone Fig 3, lane 8). Detection of siRNAs Suppression of silencing by viral proteins in plants is often associated with an inhibition of the production of small, 21 to 25 nucleotide RNAs that may be analogous to the 22 nucleotide guide RNAs identified as part of the Drosophila RISC complex [22,27,36]. We were able to identify short RNAs specific to the region of transfected dsRNA in extracts of Drosophila cells transfected with pMT/V5-His/lacZ and dsRNA (Fig 4, lane 2). A lot of larger RNA species were also detected presumably as a result of degradation of the input dsRNA. Unfortunately it is not possible to probe for the presence of short RNAs outwith the region of input dsRNA, as RNAi is not transitive in Drosophila [37]. Expression of the plant viral HC-Pro suppressor protein in the DS2-HC-Pro cells did not prevent the accumulation of these small RNAs when transfected with pMT/V5-His/lacZ and dsRNA (Fig 4, lane 4). Co-transfection of Drosophila cells with pMT/V5-His/lacZ, dsRNA and pMT-16K also resulted in production of small RNA species (Fig 4, lane 8) even though the 16K protein also partially suppresses silencing in the Drosophila cells. Possibly the inefficiency of the transfection procedure and the failure of the suppressors to completely suppress RNAi in these experiments masks any visible effect by the suppressors on the accumulation of these molecules. Similarly, small RNAs were detected in the DS2-2b cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig 4, lane 6). The dsRNA preparations used to induce RNAi were examined to determine if small RNA species of a similar size to siRNAs were present and were the species detected in figure 4. No short RNA species of 21–25 nucleotides were observed in the dsRNA preparation used to transfect the cells indicating that the species observed in the transfected cells were produced as a result of cellular processing (Fig 5, lane 3). Some larger products were observed and these are likely to have arisen as a result of premature terminations during the dsRNA synthesis reaction. Discussion Suppression of RNAi in Drosophila cells by some plant virus proteins indicates that at least part of the processes of RNAi and PTGS is conserved between plants and Drosophila. TEV HC-Pro is one of a family of proteins that suppress PTGS in plants and the CMV 2b protein has a similar ability [26]. These proteins are thought to target different components of the PTGS system, as the CMV 2b protein interferes with the spread of a silencing signal after initial induction of silencing, thus, preventing silencing from initiating in newly emerging leaves. The potyvirus HC-Pro protein, however, interferes with the maintenance of silencing in all tissues [26,27]. These differences were reflected in the Drosophila cell system where HC-Pro could suppress RNAi but the 2b gene apparently was ineffective. As there is no spread of a silencing signal in cultured Drosophila cells, the failure of the CMV 2b protein to act in this system is not unexpected. It was also significant that a mutant version of HC-Pro (K) that is defective in suppression of gene silencing [32] but is effective in proteolytic cleavage [31] was also unable to suppress RNAi in Drosophila cells. This strongly supports the idea that the TEV HC-Pro protein targets the same component of the plant PTGS system and the Drosophila RNAi system. Our demonstration that the TRV 16K protein also is able to interfere with RNAi in Drosophila cells, leads us to suggest that many other plant virus silencing suppressor proteins are likely to be functional in this system. SiRNAs were detected in our cell lines expressing HC-Pro and the TRV 16K protein even though suppression of silencing was observed. This is not totally unexpected for a number of possible reasons. Firstly, suppression of RNAi is partial in our cultures indicating that some RNAi, and presumably siRNA production, does occur in some of the cells. We have observed with cells expressing the lacZ gene alone that there is considerable variation in the amount of staining of individual cells indicating differences in the amount of gene expression in individual cells. There is no reason to suppose that this variability in gene expression is limited to the lacZ gene and it is possible that low levels of the suppressors may fail to inhibit silencing in some of the cells within a culture. Secondly, the experimental protocol requires a time delay between transfection of the cells with the lacZ plasmid and dsRNA before induction of expression of the suppressor proteins in order to allow the cells to recover from the transfection procedure. Some production of siRNAs from the dsRNA may possibly occur in cells during this lag period before the suppressors are induced. For these reasons the usefulness of the Drosophila RNAi system for studying the mechanisms of action of plant virus suppressor proteins may be limited. Mutants of Arabidopsis that are impaired in PTGS have an increased susceptibility to CMV, showing that PTGS can operate as a defensive system that targets viruses [18]. Plant virus-encoded silencing suppressors are thought to have developed as a response to this defensive aspect of the plant PTGS system, and subsequent studies have shown that, as would be expected, a wide variety of plant viruses encode silencing suppressors [24]. The suppressors encoded by these viruses are unrelated in amino acid sequence, and are likely to act at different points in the silencing process, making them ideal probes to investigate the silencing machinery. We anticipate that using different suppressors from a wide range of viruses may permit a detailed examination of the biochemical process of RNAi in Drosophila and possibly other organisms as well as allowing detailed characterisation of the mode of action of the virus suppressor proteins. Furthermore, it is becoming apparent that RNAi or PTGS may play a role in processes other than defence against foreign RNAs. Transformation of plants with TEV HC-Pro or rgs-CaM, a plant-encoded PTGS-suppressor protein related to calmodulin, interrupted normal development and led to the formation of differentiated tumours at the stem/root junction [38]. The Arabidopsis gene CARPEL FACTORY is related to Drosophila Dicer and is involved in plant development and fertility [39], and the EGO-1 gene of C. elegans also appears to have a role in both RNAi and germ-line development 15]. Components of the RNAi mechanism including a homolog of Dicer are also involved in synthesis of short temporal RNAs that regulate developmental timing in C. elegans [40-42]. Intervention with plant virus silencing suppressors may therefore have significant utility in determining the involvement of RNAi in development and differentiation in plants and animals, and possibly in manipulating these processes. Conclusions These results indicate that at least part of the process of RNAi in Drosophila and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi. Methods Plasmid constructions A region (nucleotides 1055–2449) of the TEV genome containing the HC-Pro sequence was amplified by reverse transcription – polymerase chain reaction (RT-PCR) using as a template RNA from an infected plant, and primers HC-Pro-1 (5'-CCGGTACCATGAGCGACAAATCAATCTCTGAGGC-3') and HC-Pro-2 (5'-GGCTCGAGCTACACATCTCGGTTCATCCCTCC-3'). The primers add an ATG codon to the start of the open reading frame and the HC-Pro gene was cloned as a KpnI-XhoI fragment into pMT/V5-HisA (Invitrogen) to produce plasmid pMT-HC-Pro. The same primers were also used to clone a mutant HC-Pro gene (TEV-K; [31]) from transgenic plants to produce plasmid pMT-HC-Pro/K. The 2b gene was amplified from RNA2 of CMV isolate Fny using primers 392 (5'-GAACCATGGAATTGAACGTAGGTGC) and 393 (5'-GGGTACCTCAGAAAGCACCTTCCGCC) and cloned into pGemT (Promega) before subcloning into pMT-V5-HisA. The TRV 16K gene was amplified from RNA1 of TRV isolate PpK20 using primers 433 (5'-TCATCATGACGTGTGTACTCAAGGG-3') and 434 (5'-AAGGTACCATCAAAAAGCAAACG-3') to insert BspHI and KpnI sites upstream and downstream, respectively, of the gene. The PCR product was cloned into pMT/V5-HisA to produce plasmid pMT-16K. The nucleotide sequences of the cloned PCR inserts were confirmed by sequencing. dsRNA synthesis cDNA corresponding to ~500 bp of the 5' end of the lacZ gene was amplified using pcDNA3.1/HisB/lacZ (Invitrogen) as a template and primers lacZ-1 (5'- TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGC-3') and lacZ-2 (5'- TAATACGACTCACTATAGGGCAAACGGCGGATTGACCG-3'). Both primers contain T7 RNA polymerase promoter sequences (shown underlined). The PCR product was used to direct synthesis of dsRNA using T7 RNA polymerase (Invitrogen) after which the DNA template was removed by DNase digestion. Cell culture and transfection DS2 cells, DES expression medium and the lacZ expression plasmid pMT/V5-His/lacZ were supplied as part of the Drosophila Expression System (Invitrogen) and cells were grown according to the manufacturer's instructions. Cells were grown in 60 mm dishes and transfected by calcium phosphate co-precipitation with various mixtures of 10 μg each of pMT/V5-His/lacZ and the suppressor expression plasmid DNAs and 5 μg dsRNA. In control transfections, 10 μg of an empty expression plasmid (pMT/V5-HisB) replaced the suppressor expression plasmid. After transfection the cells were washed twice in DES medium and grown for eight hours before expression of proteins was induced by addition of CuSO4 to a final concentration of 500 μM. Stably transformed lines expressing the HC-Pro, 2b or a6k genes were established by co-transfection of cells with pMT-HC-Pro or pMT-2b and pCo-Hygro (Invitrogen) followed by selection of transformed cells in medium containing hygromycin. Cells were stained 48 hrs after transfection to detect lacZ gene expression using a β-Gal Staining Kit (Invitrogen). Four randomly selected fields of view each containing ~100 cells were selected in each of duplicate plates and the number of cells staining blue was counted. Immunoblotting Cell lysates were analysed by polyacrylamide gel electrophoresis and separated proteins were transferred to nitocellulose using a carbonate buffer [43]. The blots were probed with an anti-CMV 2b [44] or anti 16K antibody [45] followed by a goat anti-rabbit alkaline phosphatase conjugate. Blots were developed using SigmaFast NBT/BCIP substrate (Sigma, Poole, UK). Northern blot analysis of RNA from Drosophila cells RNA was extracted from Drosophila cells using TriPure Isolation Reagent (Boehringer). Samples of RNA were separated by electrophoresis on formaldehyde/agarose gels, transferred to nylon membrane and probed with digoxigenin-labelled DNA probes corresponding to ~500nts at the 5' end of the lacZ gene or to the HC-Pro cDNA described above, as appropriate and essentially as described [46]. The probes were made by PCR using primers lacZ-3 (5'- GGAGACCCAAGCTGGCTAGC-3') and lacZ-4 (5'- GGCAAACGGCGGATTGACCG-3') for the lacZ probe, and HC-Pro-1 and HC-Pro-2 for the HC-Pro probe. Digoxigenin-labeled RNA Molecular Weight Marker II (Roche Molecular Biochemicals) was run as a size marker. For detection of short (~23nt) RNA species total RNA preparations from Drosophila cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad), following the manufacturers instructions. ~20 μg of the short RNA species were separated by electrophoresis in a 15% polyacrylamide gel containing 7 M Urea and electroblotted as described by Llave et al. [27]. After electrophoresis the gel was stained with ethidium bromide and photographed under ultra-violet light before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. For the detection of siRNAs, a digoxigenin-labelled RNA probe complementary to the 5' region of the lacZ gene as described above was synthesised from a PCR fragment containing the gene fragment downstream of a T7 RNA polymerase promoter, and hybridised to the blots according to the manufacturer instructions (Roche Diagnostics). Induction and detection of siRNAs to GFP or to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto et al. [47]. Competing interests None declared. Authors' contributions BR conceived of the study, and participated in its design, carried out the Drosophila expression experiments, northern blots and drafted the manuscript. SD carried out the immunoassays. TC carried out some of the siRNA assays. SM participated in the design of the study, carried out some of the siRNA assays and contributed to the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Hugh Barker for useful discussions and help with photography of cells. We thank Vicki Vance for providing us with TEV HC-Pro mutant K. SCRI is grant-aided by the Scottish Executive Rural affairs Department. Figures and Tables Figure 1 Expression of plant virus silencing suppressor genes in Drosophila cells. A. Immunoblot of lysates from control cells (lanes 1,2 and 5,6), cells transiently transfected with pMT-2b (lanes 3,4) or cells stably transformed with pMT-2b (lanes 7,8). Cells represented in lanes 2, 4, 6 and 8 were induced by addition of CuSO4 and cells represented in lanes 1, 3, 5 and 7 were uninduced. Lanes 9 and 10 were lysates from control plant or plant infected with CMV respectively. Lysates were analysed by polyacrylamide gel electrophoresis, proteins transferred to nitrocellulose by electroblotting and probed with an anti-2b antibody. The position of migration of a SeeBlue Plus 2 Pre-stained molecular weight marker (Invitrogen) band is shown. B. Northern blot analysis of RNA from control cells (lane 1) or cells transfected with pMT-HC-Pro (lane 2) or with pMT-HC-Pro/K (lane 3). Total cell RNA was separated on 1% agarose gels, transferred to nylon membrane and probed with an HC-Pro specific probe. The position of migration of a digoxigenin-labeled RNA molecular weight marker (Roche Molecular Biochemicals) band is shown. C. Northern blot analysis of RNA from DS2-HC-Pro cells (lane 1) or control cells (lane 2). RNA was examined as above. The position of migration of a digoxigenin-labeled RNA molecular weight marker band is shown. Figure 2 Expression of TRV 16k protein in Drosophila cells. Immunoblot of lysates from control cells (lanes 1,2 and 5,6), cells transiently transfected with pMT-16k (lanes 3,4) or cells stably transformed with pMT-16k (lanes 7,8). Cells represented in lanes 2, 4, 6 and 8 were induced by addition of CuSO4 and cells represented in lanes 1, 3, 5 and 7 were uninduced. Lanes 9 and 10 were lysates from control plant or plant infected with TRV respectively. Lysates were analysed by polyacrylamide gel electrophoresis, proteins transferred to nitrocellulose by electroblotting and probed with an anti-16k antibody. The position of migration of SeeBlue Plus 2 Pre-stained molecular weight markers (Invitrogen) bands is shown. Figure 3 Northern blot analysis of lacZ RNA in DS2 cells. Upper Panels. Northern blot analysis of total cellular RNA probed with a lacZ-specific probe corresponding to ~500 bp of the 5' end of the lacZ gene. RNA was extracted from DS2 cells (lanes 1, 2, 7 and 9), DS2-HC-Pro cells (lanes 3 and 4), DS2-16k cells (lanes 5 and 6) or DS2-2b cells (lanes 8 and 10). All cells were transfected with pMT/V5-His/lacZ and lanes 2, 4, 6, 9 and 10 were co-transfected with pMT/V5-His/lacZ and dsRNA. The lacZ specific band migrated between 2661nt and 4742 nt digoxigenin labelled RNA size markers. Lower Panels – Stained gels showing loadings of Drosophila rRNA before blotting. Figure 4 Detection of small lacZ gene-specific RNA. Total RNA was isolated from Drosophila cell cultures and fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels and stained with ethidium bromide before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. The membrane was probed with an antisense digoxigenin-labelled lacZ transcript corresponding to ~500 nt at the 5' end of the lacZ gene. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells (lanes 1, 2), DS2-HC-Pro cells (lanes 3, 4), DS2-2b cells (lanes 5, 6) and DS2-16k cells (lanes 7, 8). Cells represented in lanes 1, 3, 5 and 7 were transfected with pMT/V5-His/lacZ and those represented in lanes 2, 4, 6 and 8 were transfected with pMT/V5-His/lacZ and dsRNA. The line to the right indicates the position of GFP specific siRNAs produced in plants as described by Canto et al. (47). The lower panel shows the RNA species detected by staining of the gel before electroblotting. Figure 5 Small lacZ gene-specific RNA is not present in the dsRNA preparation used to induce RNAi. RNA preparations from Drosophila cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels, and electroblotted. A digoxigenin-labelled RNA probe complementary to the 5' region of the lacZ gene was used for the detection of siRNAs in lanes 1–3. Induction and detection of siRNAs to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto et al. (47) and were used as markers in lanes 4 and 5. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells transfected with pMT/V5-His/lacZ (lane 1) or non-transfected DS2 cells (lane 2) or in the dsRNA preparation used to transfect DS2 cells to induce RNAi (lane 3). Also shown are the small RNA species present in plant tissue agroinfiltrated with a binary vector (lane 5) or in non-agroinfiltrated tissue (lane 4) detected using a digoxigenin-labelled RNA probe to 35S promoter sequences present in the binary vector as markers. The positions of migrations of the small (~21nt) RNAs are indicated by arrows. Table 1 Effects of plant virus gene silencing suppressors on RNAi in Drosophila cells. Cell Type Transfection % cells stained * DS2 pMT/V5-His/lacZ 82.86 ± 9.12 DS2 pMT/V5-His/lacZ + dsRNA 17.70 ± 4.25 DS2 pMT/V5-His/lacZ + dsRNA + pMT-HC-Pro 45.78 ± 4.89 DS2 pMT/V5-His/lacZ + dsRNA + pMT-HC-Pro-K 14.62 ± 2.81 DS2 pMT/V5-His/lacZ + dsRNA + pMT-2b 11.13 ± 1.58 DS2 pMT/V5-His/lacZ + dsRNA + pMT-16K 47.25 ± 3.4 DS2-HC-Pro pMT/V5-His/lacZ 42.04 ± 3.07 DS2-HC-Pro pMT/V5-His/lacZ + dsRNA 31.70 ± 2.46 DS2-scAb S20 pMT/V5-His/lacZ 40.67 ± 2.36 DS2-scAb S20 pMT/V5-His/lacZ + dsRNA 6.50 ± 1.2 DS2-2b pMT/V5-His/lacZ 41.16 ± 2.36 DS2-2b pMT/V5-His/lacZ + dsRNA 9.17 ± 2.56 * Four randomly selected fields of view each containing ~100 cells were selected in each of duplicate plates and the number of cells staining blue was counted for each experiment. The figures shown are the means from four experiments for the DS2 cells and three experiments for the DS2-HC-Pro and DS2-ScAb S20 cells. ==== Refs Hannon GJ RNA Interference Nature 2002 418 244 251 12110901 10.1038/418244a Cerutti H RNA Interference: travelling in the cell and gaining functions? Trends in Genetics 2003 19 39 46 12493247 10.1016/S0168-9525(02)00010-0 Kennerdell JR Carthew RW Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway Cell 1998 95 1017 1026 9875855 10.1016/S0092-8674(00)81725-0 Fire A Xu S Montgomery MK Kostas SA Driver SE Mello CC Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 1998 391 806 811 9486653 10.1038/35888 Wargelius A Ellingsen S Fjose A Double-stranded RNA induces specific developmental defects in zebrafish embryos Biochem Biophys Res Comm 1999 263 156 161 10486270 10.1006/bbrc.1999.1343 Wianny F Zernicka-Goetz M Specific interference with gene function by double-stranded RNA in early mouse development Nat Cell Biol 2000 2 70 75 10655585 10.1038/35000016 Elbashir SM Harborth J Lendeckel W Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 2001 411 494 498 11373684 10.1038/35078107 Waterhouse PM Wang M-B Lough T Gene silencing as an adaptive defence against viruses Nature 2001 411 834 842 11459066 10.1038/35081168 Cogoni C Homology-dependent gene silencing mechanisms in fungi Ann Rev Microbiol 2001 55 381 406 11544361 10.1146/annurev.micro.55.1.381 Cogoni C Macino G Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase Nature 1999 399 166 169 10335848 10.1038/20215 Cogoni C Macino G Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase Science 1999 286 2342 2344 10600745 10.1126/science.286.5448.2342 Catalanotto C Azzalin G Macino G Cogoni C Gene silencing in worms and fungi Nature 2000 404 245 10749199 10.1038/35005169 Ketting RF Haverkamp THA van Leunen HGAM Plasterk RHA mut-7 of C-elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNAse D Cell 1999 99 133 141 10535732 10.1016/S0092-8674(00)81645-1 Tabara H Sarkissian M Kelly W Fleenor J Grishok A Timmons L Fire A Mello C The rde-1 gene, RNA interference, and transposon silencing in C. elegans Cell 1999 99 123 132 10535731 10.1016/S0092-8674(00)81644-X Smardon A Spoerke JM Stacey SC Klein ME Mackin N Maine EM EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C-elegans Curr Biol 2000 10 169 78 10704412 10.1016/S0960-9822(00)00323-7 Dalmay T Hamilton H Rudd S Angell S Baulcombe DC SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. Cell 2000 101 543 553 10850496 10.1016/S0092-8674(00)80864-8 Fagard M Boutet S Morel J-B Bellini C Vaucheret H AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals Proc Natl Acad Sci USA 2000 97 11650 11654 11016954 10.1073/pnas.200217597 Mourrain P Beclin C Elmayan T Feuerbach F Godon C Morel JB Jouette D Lacombe AM Nikic S Picault N Remoue K Sanial M Vo TA Vaucheret H Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance Cell 2000 101 533 542 10850495 10.1016/S0092-8674(00)80863-6 Caplen NJ Fleenor J Fire A Morgan RA Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems Gene 2000 252 95 105 10903441 10.1016/S0378-1119(00)00224-9 Hammond SM Bernstein E Beach D Hannon GJ An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells Nature 2000 404 293 296 10749213 10.1038/35005107 Ui-Tei K Zenno S Miyata Y Saigo K Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target FEBS Lett 2000 479 79 82 10981711 10.1016/S0014-5793(00)01883-4 Bernstein E Caudy AA Hammond SM Hannon GJ Role for a bidentate ribonuclease in the initiation step of RNA interference Nature 2001 409 363 366 11201747 10.1038/35053110 Vance V Vaucheret H RNA silencing in plants – defense and counterdefense Science 2001 292 2277 2280 11423650 10.1126/science.1061334 Voinnet O Pinto YM Baulcombe DC Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants Proc Natl Acad Sci USA 1999 96 14147 14152 10570213 10.1073/pnas.96.24.14147 Kasschau KD Cronin S Carrington JC Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase Virology 1997 228 251 262 9123832 10.1006/viro.1996.8368 Brigneti G Voinnet O Li WX Ji LH Ding SW Baulcombe DC Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana EMBO J 1998 17 6739 6746 9822616 10.1093/emboj/17.22.6739 Llave C Kasschau KD Carrington JC Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway Proc Natl Acad Sci USA 2000 97 13401 13406 11078509 10.1073/pnas.230334397 Guo HS Ding SW A viral protein inhibits the long range signalling activity of the gene silencing signal EMBO J 2002 21 398 407 11823432 10.1093/emboj/21.3.398 Voinnet O Lederer C Baulcombe DC A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana Cell 2000 103 157 167 11051555 10.1016/S0092-8674(00)00095-7 Li H Li XL Ding SW Induction and Suppression of RNA Silencing by an Animal Virus Science 2002 296 1319 1321 12016316 10.1126/science.1070948 Shi XM Miller H Verchot J Carrington JC Vance VB Mutations in the region encoding the central domain of helper component-proteinase (HC-Pro) eliminate potato virus X/potyviral synergism Virology 1997 231 35 42 9143300 10.1006/viro.1997.8488 Anandalakshmi R Pruss GJ Ge X Marathe R Mallory AC Smith TH Vance VB A viral suppressor of gene silencing in plants Proc Natl Acad Sci USA 1998 95 13079 13084 9789044 10.1073/pnas.95.22.13079 Reavy B Ziegler A Diplexcito J Macintosh SM Torrance L Mayo M Expression of functional recombinant antibody molecules in insect cell expression systems Protein Expression & Purification 2000 18 221 228 10686153 10.1006/prep.1999.1191 MacFarlane SA Molecular biology of the tobraviruses J Gen Virol 1999 80 2799 2807 10580040 Liu H Reavy B Swanson M MacFarlane SA Functional replacement of the Tobacco rattle virus cysteine-rich protein by pathogenicity proteins from unrelated plant viruses Virology 2002 298 232 239 12127786 10.1006/viro.2002.1421 Mallory AC Ely L Smith TH Marathe R Anandalakshmi R Fagard M Vaucheret H Pruss G Bowman L Vance VB HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal Plant Cell 2001 13 571 583 11251097 10.1105/tpc.13.3.571 Roignant J-Y Carré C Mugat B Szymczak D Lepesant Antoniewski C Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. RNA 2003 9 299 308 12592004 10.1261/rna.2154103 Anandalakshmi R Marathe R Ge X Herr Jr JMMau C Mallory A Pruss G Bowman L Vance VB A Calmodulin-Related Protein That Suppresses Posttranscriptional Gene Silencing in Plants Science 2000 290 142 144 11021800 10.1126/science.290.5489.142 Jacobsen SE Running MP Meyerowitz EM Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems Development 1999 126 5231 5243 10556049 Grishok A Pasquinelli AE Conte D Li N Parrish S Ha I Baillie DL Fire A Ruvkun G Mello CC Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing Cell 2001 106 23 34 11461699 10.1016/S0092-8674(01)00431-7 Hutvāgner G McLachlan J Pasquinelli AE Bālint E Tuschl T Zamore PD A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA Science 2001 293 834 838 11452083 10.1126/science.1062961 Ketting RF Fischer SEJ Bernstein E Sijen T Hannon GJ Plasterk RHA Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans Genes Develop 2001 15 2654 2659 11641272 10.1101/gad.927801 Dunn SD Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on western blots by monoclonal antibodies Anal Biochem 1986 157 144 153 3532863 Mayers CN Palukaitis P Carr JP Subcellular distribution analysis of the cucumber mosaic virus 2b protein J Gen Virol 2000 81 219 226 10640561 Liu DH Robinson DJ Duncan GH Harrison BD Nuclear location of the 16K nonstructural protein of tobacco rattle virus J Gen Virol 1991 72 1811 1817 1831493 Webster KD Barker H Foster GD Detection and quantification of transcript RNA in transgenic plants using digoxigenin-labelled cDNA probes In Methods in Molecular Biology: Plant Virology 1998 20 Totowa, NJ: Humana Press 437 445 Canto T Cillo F Palukaitis P Generation of siRNAs by T-DNA sequences does not require active transcription or homology to sequences in the plant Mol Plant-Microbe Interact 2002 15 1137 1146 12423019

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==== Front BMC Med Inform Decis MakBMC Medical Informatics and Decision Making1472-6947BioMed Central London 1472-6947-4-121533933610.1186/1472-6947-4-12Research ArticleWireless local area network in a prehospital environment Chen Dongquan [email protected] Seng-jaw [email protected] Gary J [email protected] Helmuth F [email protected] Health Informatics Program, Department of Health Services Administration School of Health Related Professions. University of Alabama at Birmingham (UAB). Birmingham, Alabama, USA2 Current Address: Biostatistics and Bioinformatics Unit, Comprehensive Cancer Center of UAB. Birmingham, Alabama, USA3 Department of Electrical & Computer Engineering, Centre for Telecommunications of UAB. Birmingham, Alabama, USA2004 31 8 2004 4 12 12 2 12 2003 31 8 2004 Copyright © 2004 Chen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Wireless local area networks (WLANs) are considered the next generation of clinical data network. They open the possibility for capturing clinical data in a prehospital setting (e.g., a patient's home) using various devices, such as personal digital assistants, laptops, digital electrocardiogram (EKG) machines, and even cellular phones, and transmitting the captured data to a physician or hospital. The transmission rate is crucial to the applicability of the technology in the prehospital setting. Methods We created two separate WLANs to simulate a virtual local are network environment such as in a patient's home or an emergency room (ER). The effects of different methods of data transmission, number of clients, and roaming among different access points on the file transfer rate were determined. Results The present results suggest that it is feasible to transfer small files such as patient demographics and EKG data from the patient's home to the ER at a reasonable speed. Encryption, user control, and access control were implemented and results discussed. Conclusions Implementing a WLAN in a centrally managed and multiple-layer-controlled access control server is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management. ==== Body Background The development of the Internet has encouraged doctors to use computers and hospitals to use wireless communications [1], since wireless technology offers many benefits over its wired counterpart, including ease of installation and access to network information [2-6], and higher productivity and convenience [2]. One study using personal digital assistants (PDAs) connected to a network showed that the device was of limited use in transmitting data in prehospital stroke management [7]. Another study showed that cellular phones, pagers, or other radio-based devices will remain an important communication mode in the near future [8]. The advancement of wireless local area network (WLAN) technology provides the potential to allow physicians to obtain a patient's information anywhere, even before the patient reaches the emergency room (ER) (Orthner, personal communication). Timely access to a patient's information may fundamentally improve patient care [9] in both pre- and in-hospital settings, due to earlier doctor interventions. At present, patient data such as electrocardiograms (EKG) and demographics are seldom sent from the prehospital environment to the ER before the ambulance arrives [10]. As a result, some preventive measures have to be given, regardless of need (e.g., aspirin [11] or thrombolysis [12] for presumed acute myocardial infarction. However, despite the potential benefits of wireless technology in prehospital settings, the application of this technology has been slow and few related studies have been carried out. The objective of this study was to assess the ability of wireless technology to facilitate data communication between a prehospital setting and an ER (Orthner, personal communication). The idea was for all the data collected by paramedical personnel to be transmitted to an ER server from the patient's home, on the way to the ER, or upon arrival at the ER. Thus the transmission rate is crucial to the usefulness and applicability of the technology. To test the feasibility of wireless data transmission under the various scenarios, two separate WLANs were created, one around our office and another in a house. In this report, we discuss our testing of the wireless technology, and its potentials and limitations in simulated prehospital settings. Methods The WLAN products used (Aironet 340 and 350 series wireless client adaptors and access points (APs); Cisco) offered 11-megabits-per-second (Mbps) transmission rates, built-in security features (including 40- to 128-bit encryption) and Web-based management. The transmission rates of files of different sizes were measured with different APs, patch antennae, clients, and transfer methods. The security of patient data was ensured using a centrally managed Access Control Server (ACS). Other issues such as standards, roaming, and cost are also discussed here. Within a WLAN, data are transmitted between a server and its wireless clients via an AP antenna. Both workstations and laptops were used here as servers, and file transfer rates were measured for both systems. We used Gateway Select series, Dell Inspiron series, and a Toshiba Satellite laptop computer as clients. The computers had CPU operating at 0.8–1.2 GHz, 256–1024 MB of RAM, and 10–40 GB hard drives, and all ran the Microsoft Windows 2000 Professional operating system. PDAs (Ipaq Pocket PCs, models 3550 and 3570, 200-MHz CPU, 32–64 MB RAM, Compaq) were also tested as wireless clients. The wireless coverage was tested using two APs (Aironet 340 and 350 series, Cisco) and a patch antenna (S2406P, Cushcraft Corporation). The feasibility of using cellular phones (StarTac 7868, Motorola) in data transmission in the area not covered by the WLAN was also tested. The various software used in the study for wireless client management, file transfer, and access control included the Aironet Client Utility (Cisco), Link Status Meter (LSM, Cisco), the ACS (V3.0, Cisco), Phone Tools (BVRP Software) for faxing, and file transfer protocol (FTP) for measuring the file transmission rate. The software LSM classifies the link status as the percentage of maximum signal strength and quality: "excellent" (>75%), "good" (40–75%), "fair" (20–40%), or "poor" (<20%); where signal strength and quality refer to the client adapter's radio signal at the time packets are being received, quantified as bytes transmitted and received and the errors that occur. Detailed descriptions of the mentioned software are available from the manuals provided by the vendors. The WLAN and its configuration The Aironet 340 and 350 series APs were tested by a two-step approach. In the first step, one AP was connected directly with the server that was not connected to the campus Ethernet backbone. In the second step, the AP was assigned a public Internet Protocol (IP) address and connected to the Ethernet backbone in the Susan Mott Webb Nutrition (Webb) Building at the University of Alabama at Birmingham. The IP address was assigned to an AP through either HyperTerminal or a Web console using a Web browser. An administrator ID and password were then created to enhance the Web console security. The client computer required a type II PCMCIA (Personal Computer Memory Card International Association) card slot. Every client needed a functional IP address to become associated with the AP. The Wired Equivalent Privacy (WEP) keys were enabled for both the AP and the clients to ensure two-way authentication. Comparison of different coverage of APs and patch antennas The coverage of the WLAN was found to highly structure dependent. The floor of the Webb building measures about 60 by 25 meters. A single Aironet 340 AP was unable to cover the entire floor with a "good" link status. This was achieved using two (more powerful) Aironet 350 APs. Achieving the "excellent" link status on the floor required the use of the S2406P (Cushcraft) patch antenna. The wireless clients associated with the AP had a "fair" or "good" status one floor up and one floor down from the floor where the AP was located. There was a small area outside the 5-floor building in which the clients could associate with the AP with a "poor" status. Results Link statuses around a simulated patient's home In order to test the feasibility of implementing an isolated LAN around a patient's home, we chose a two-story house with the layout shown in Figure 1. Two simulated scenarios were tested: one with an ambulance parked on the street next to the house (Figure 1A and 1C), and the other with the ambulance parked next to the house in its parking lot (Figure 1B and 1D). Two connection modes under each scenario were also tested: one mode used the AP-client connection (Figure 1A and 1B) and the other used a peer-to-peer connection without the AP (Figure 1C and 1D). The link statuses both inside and outside the house were at least "good", and some of the area close to the AP in both scenarios had "excellent" coverage. This suggested that an ambulance with a patch antenna could communicate at a reasonable rate with handheld devices operating inside the patient's home, through either an AP or a direct peer-to-peer connection. File transfer rate with laptops To quantify the baseline signal strength, one Aironet 350 AP was tested in the open area: "excellent", "good", "fair", and "poor" link statuses were obtained within 10, 25, 50, and 100 feet (~4, 10, 20, and 39 meters), respectively. As shown in Table 1A, the file transfer rate for a 10-MB file was 224–2,000 kbps, depending on the distance. Interestingly, higher rates were reached for files of size 10–100 MB. The different methods and directions of file transfer might affect the rate. Methods such as FTP transfer, copying and pasting between folders, and Microsoft Briefcase synchronization were tested. Other factors that may affect transmission were also tested, including the initiation direction (pulling or pushing, in terms of the choice of client and server; see below) and relative physical motion. One Gateway workstation and two Inspiron 4000 laptops were used. To simplify the test, a single medium-sized file (50 MB) was chosen for the purpose. We chose a 50-MB file since it corresponds to a typical high-quality EKG image. As indicated in Table 1B, pushing refers to a transfer from the server to clients if initiated from the server side, and from a client to the server if initiated from the client side; whereas pulling refers to the transfer from the server to clients if initiated from the client side, and from a client to the server if initiated from the server side. Pulling a file led to a higher rate of transfer, in all link statuses (i.e., distances). The speed was lower when two clients transferred the 50-MB file at the same time, and it was generally lower for file transfer between two clients than between a server and a client (Table 1B, Sections III-IV). As expected, the transfer rate was slower while the client device was moving (as shown in Table 2B, section V). However, it was still found that in a WLAN environment, paramedics carrying data-collecting devices could move around and transmit data by different methods and directions at a reasonable rate. The simultaneous file transfer that involves multiple clients/users is a more likely scenario in real emergency settings. Under a "good" link status, the ability of five laptop computers to pull a single file from the FTP server was tested both individually (Table 2A) and simultaneously (Table 2B). The data rate reached 5.9 Mbps when a single client was transferring, and fell to below 2 Mbps when multiple clients were involved simultaneously (based on four independent tests). This lower rate, however, is still within a reasonable range, considering the file size. File transfer rate with Pocket PC and cellular phone We chose Ipaq Pocket PCs as PDAs due to their relatively large amount of RAM compared to other PDAs, and a cellular phone (Motorola) as alternative data transmission device when the PDA was outside the WLAN coverage (to simulate the scenario when a long-range antenna, such as a yagi antenna (Cisco), is not available). The transfer rates for single files of different sizes are summarized in Table 3A. The faxing speed through the phone did not appear to be correlated with the file size, since a 50-fold difference in file size resulted in a 30% difference in the time needed to complete the file transmission. The reason is unknown, and should be further investigated. Enhancing the WLAN security using an ACS A Cisco V3 ACS was used to improve the security of the network [13]. As shown in Figure 2, the Active Directory facility of a Windows 2000 Server was used as a network access server to communicate with the ACS for authentication, authorization, and accounting (AAA) [14]. The ACS was able to control client access to the network through the AAA process. A private local area network When managing a WLAN with many clients, there are often insufficient public IP addresses. The solution is to use either the Dynamic Host Configuration Protocol (DHCP) or a private LAN with a Network Address Translation (NAT) server. DHCP services are not easily managed and sometimes create security challenges to network administrators in determining the user's identity. A private LAN seems to be a better solution and more applicable in most ER environments where many wireless clients may transmit at same times, thus maintaining relatively high-speed connections. Figure 3 shows a private LAN with a NAT server that was configured and tested. All wireless clients were centrally managed through an ACS server. The file transfer rates were similar to those achieved with public IP addresses under similar conditions (data not shown). Cost of a small-scale WLAN Health-care organizations have traditionally been slow in accepting WLAN technology in clinical practice. One of the major concerns has been its cost [15,16], followed by security [17-20], although the benefits of WLANs have been demonstrated in many fields including telemedicine [21]. The cost of a simple WLAN like the one tested here was calculated (Table 4). Discussion Synchronization After collecting all patient data at a patient's home, the data must ultimately be transferred to the ER. This involves two critical synchronization steps: (1) from the patient's home to a server on the ambulance, and (2) from the ambulance to the ER (while in transit or upon arrival). Here the WLAN was employed for both of these steps, using Microsoft Briefcase and Windows Workgroups. Automatic synchronization with the destination server and batch synchronization were desired. The ultimate objective is, however, to link the two synchronization steps using a long-range antenna that reaches up to 25 miles (e.g., a yagi antenna from Cisco). This would significantly shorten the time needed to transfer data from a patient's home to the ER, since the data will reach a ER WLAN earlier. We are currently performing the associated experiments. We also tested the use of a cellular phone and other types of PDA (e.g., a Palm Pilot) with network capabilities in transmitting a small (up to 50 MB) but critical file. The results suggested that cellular phones or PDAs with network cards can be effective alternatives to the use of a long-range antenna to transmit data from a patient's home to the ER. Wireless transfer of EKG data EKG data are considered very valuable in the early detection, early intervention, and possibly better outcome of heart attack patients [9]. The use of a wearable device with sensors to monitor specific physiological signals and communicate with a personal server has been reported [22]. Land-based telephone lines have also been employed to transmit EKG data and for monitoring by clinical personnel [23]. In our study, we showed that files up to 5–10 MB (the size of a typical high-quality digitalized EKG image) could be transferred using FTP or other file transfer methods within minutes. Handheld devices such as a Pocket PC and cellular phone may be useful in transmitting EKG files when the ambulance is still at the patient's home, as shown in Table 3. The time required to transmit a file is proportional to its size in the case of a Pocket-PC-to-laptop transfer, but this was found not to be the case between a cellular phone and a fax machine. The reason for this discrepancy is unknown, and needs to be further investigated. The use of a long-range antenna may ultimately be needed to increase the transmission capacity and speed. Interferences The WLAN operates at 2.42 GHz with an output power of 100 mW, which may pose a risk of interference with medical devices using similar frequencies. Previous studies have shown that a WLAN may interfere with medical devices in close proximity [24] but is unlikely to be interfered with by such devices [25]. Further studies are needed to clearly address the possibility. In another study, infrared modems exhibited a similar performance to a wired system even in an electrically noisy environment [26], indicating that infrared wireless connectivity can be safely and effectively used in operating rooms. These studies suggest that a WLAN can be acceptable for use in prehospital settings if careful interference testing is conducted. Security and privacy The major concern over a WLAN is its security [17-20], especially when personal information is involved. It has been reported that the open-air clear-text transmission of WEP keys and MAC addresses increases network vulnerability [13,27-29]. One approach to minimizing the risk is to control the access of remote and/or wireless clients through the Remote Access Dial-in User Service and AP management using the Extensible Authentication Protocol. The regulation by the Healthcare Insurance Portability and Accountability Act may further delay an organization's decision to adopt WLAN technology, although both the Institute of Electrical and Electronic Engineering (IEEE) 802.3 and the OpenAir standard specifications offer security protection (these are the two major standards in the unlicensed commercial 2.4-GHz WLAN market). According to our experiences, the following steps are required to implement a secure WLAN. First, anonymous access should be disabled and the Service Set Identifier of an AP and data encryption key (WEP key) should be enabled. Secondly, a Web console should be used to designate an administrator and manage APs and clients. Thirdly, an ACS server such as Cisco Secure ACS should be implemented to work with Active Directory in order to offer both device- and user-dependent AAA services. Digital certificates should be applied whenever possible for mutual authentication to protect sensitive information through secure server access and secure Web access. In addition, the physical security of the APs, client, and server computers can never be overemphasized. Standards and interoperability The IEEE 802.11 specification addresses both the physical and MAC layers (Orthner, personal communication), and the OpenAir 2.4 interface standard is derived from the Wireless LAN Interoperability Forum [29] and needs to be interoperable with the IEEE 802.11 standards. The 5-GHz band WLAN standard (IEEE 802.11a) will become more popular once its cost decreases and the required components become more widely available. The use of standardized compliant devices facilitates communication and interoperability. Limitations of the study The present study was mainly based on the Windows operating system and Cisco wireless products. IEEE 802.11a products for the next generation of WLANs are emerging quickly from various vendors. Hence the stability, compatibility, and interoperability with other vendors require further evaluation. Although currently it is relatively expensive to implement a WLAN using this new protocol, the prices and capabilities are expected to improve within the near future. Conclusions Application of WLAN technology will help both paramedics and other health-care professionals in their daily acquisition of information in a localized area such as within a patient's home, an office, a small clinic, or an ER. Implementing a WLAN in a centrally managed and multiple-layer-controlled ACS is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management. Competing interests None declared. Authors' contributions D. Chen, the principal investigator, was most involved in conducting the experiments. H.F. Orthner participated in data collection in the simulated patient's home. H.F. Orthner was the sponsor and S.-J. Soong and G.J. Grimes were the advisors of the fellowship awarded to D. Chen from the National Library of Medicine, National Institute of Health, and they contributed significantly to the design, coordination, and performing of experiments. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements This project has been funded in part with US federal funds from the National Library of Medicine, National Institute of Health, under Contract No. N01-LM-0-3524 and under Fellowship No. F38LM07185. Figures and Tables Figure 1 An aerial view of the use of a patch antenna at a simulated patient's home. The AP – clients connection (A and B) and peer-to-peer connection (C and D) configurations were tested. The arrows indicate the locations and the beaming directions of the APs with a patch antenna. The stars indicate the locations of the wireless devices in the peer-to-peer connections. The link statuses were measured as described in the Methods: the solid and dashed circles indicate "excellent" and "good" statuses, respectively. Panel A: Simulated ambulance parked on the street. Panel B: Simulated ambulance parked in the parking lot of the house. C and D: Peer-to-peer connection was created between two wireless clients. Figure 2 The Cisco Secure ACS and its configuration. The AP was configured to use ACS AAA services for the Extensible Authentication Protocol (EAP)-enabled wireless devices over the WLAN. A Windows 2000 Server running Active Directory (AD) was used to mimic a network access server (NAS) to negotiate with ACS through the Remote Access Dial-in User Service (RADIUS) protocol. The NAS had an enabled RAS connection for the ACS. Figure 3 A private WLAN with an ACS. A workstation running the Linux operating system functioned as a Network Address Translation (NAT) server. The private LAN was linked with the Internet through a switched hub or router. The AP was configured to use ACS for AAA services for the EAP-enabled wireless devices over the WLAN. A Windows 2000 Server running Active Directory was used to mimic a NAS to negotiate with ACS through the RADIUS protocol. Table 1 File transfer directions and rates (kbps, mean ± SD, n = 4) A. File transfer rate in an open area between two laptops for different file sizes and link statuses 1 MB 10 MB 100 MB 1000 MB Excellent 400 2000 1660 980 Good 210 890 1110 830 Fair 90 660 740 660 Poor 60 220 250 340 B. Transfer rates for a 50-MB file between a server and clients under different link statuses I. Server to one client Pushing Pulling Excellent 3090 ± 70 5540 ± 180 Good 2870 ± 280 5530 ± 130 Fair 630 ± 500 2070 ± 550 II. One client to server Pushing Pulling Excellent 2600 ± 110 5210 ± 410 Good 2710 ± 330 4350 ± 1030 Fair 340 ± 80 750 ± 440 III. Two clients to server Client 1 Client 2 Excellent 2160 ± 60 3130 ± 320 Good 2140 ± 370 3230 ± 40 IV. Client 1 to client 2 Excellent 2400 ± 70 Good 2560 ± 130 V. Server to one client while moving Excellent 2500 ± 500 Good 1200 ± 410 Table 2 Rate of file transfers involving multiple clients (kbps, mean ± SD, n = 4) A. Transferring files individually Client 50 MB Gateway Solo 5830 ± 1340 Toshiba Satellite 350 5250 ± 600 Inspiron 8000 5260 ± 990 Inspiron 4000-1 4890 ± 490 Inspiron 4000-2 5960 ± 910 B. Transferring files simultaneously Client 50 MB 40 MB 30 MB Gateway Solo 1330 ± 180 1430 ± 140 1740 ± 490 Toshiba Satellite 350 1600 ± 340 1580 ± 160 1610 ± 240 Inspiron 8000 1400 ± 190 1470 ± 140 1500 ± 130 Inspiron 4000-1 1490 ± 200 1580 ± 240 1590 ± 260 Inspiron 4000-2 1240 ± 440 1540 ± 190 1560 ± 280 20 MB 10 MB 1 MB Gateway Solo 1690 ± 200 1900 ± 400 7140 ± 110 Toshiba Satellite 350 1850 ± 60 1860 ± 350 6830 ± 120 Inspiron 8000 1730 ± 170 1820 ± 220 4360 ± 2800 Inspiron 4000-1 1690 ± 190 2000 ± 470 3780 ± 1060 Inspiron 4000-2 1840 ± 310 1770 ± 400 3160 ± 1400 Table 3 Pocket PC and cellular phone file transfers (SD: 10~25%, n = 4) A. Pocket PC to laptop B. Cellular phone to a fax machine File size (kB) Seconds File size (kB) Seconds 10 <1 10 134 100 2 100 155 1000 15 500 166 5000 150 1500 180 Table 4 Cost of a WLAN of similar scale to the one implemented here Component No. needed Unit price (US $) Total US$ Workstation and accessories 1 2500 2500 Laptop and accessories 2 2500 5000 Pocket PC and accessories 3 600 1800 Palm Pilot and accessories 2 500 1000 APs 2 400 800 Patch antennas 2 200 400 Wireless adaptors 5 300 1500 Cellular phone and network costs 1 250 250 Total (US $) 13250 Note: software costs are usually associated with the purchase of hardware and not included. ==== Refs Goldstein D Toth CL MedDigital trends and tactics to lead into the future Physician Exec 2001 27 20 8 11291217 Levine R Avoid the paper chase. Real-time, wireless patient documentation helps therapists save both time and money Healthc Inform 1999 16 73 4 10345769 Pinkert J Contemporary issues in HIM. Kerberos – security for sensitive data J Ahima 1994 65 22 4 26-8; quiz 56-7 10134105 Pinkert J Contemporary issues in HIM. Wireless LANs J Ahima 1994 65 28 34 quiz 36, 38 10134093 Lovell N Celler BG Implementation of a clinical workstation for general practice Medinfo 1995 8 777 8591325 Lovell N Magrabi F Celler BG Huynh K Garsden H Web-based acquisition, storage, and retrieval of biomedical signals IEEE Eng Med Biol Mag 2001 20 38 44 11446208 10.1109/51.932723 Quaglini S Caffi E Boiocchi L Panzarasa S Cavallini A Micieli G Web-based Data and Knowledge Sharing Between Stroke Units and General Practitioners Proc AMIA Symp 2003 534 8 14728230 Heslop L Howard A Fernando J Rothfield A Wallace L Wireless communications in acute health-care J Telemed Telecare 2003 9 187 93 12952687 10.1258/135763303322225490 Garrett P Boyd SY Bauch TD Rubal BJ Bulgrin JR Kinkler ES Feasibility of real-time echocardiographic evaluation during patient transport J Am Soc Echocardiogr 2003 16 197 201 12618725 10.1067/mje.2003.16 Anantharaman V Swee Han L Hospital and emergency ambulance link: using IT to enhance emergency pre-hospital care Int J Med Inf 2001 61 147 61 11311669 10.1016/S1386-5056(01)00137-X Barbash I Freimark D Gottlieb S Hod H Hasin Y Battler A Crystal E Matetzky S Boyko V Mandelzweig L Behar S Leor J Outcome of myocardial infarction in patients treated with aspirin is enhanced by pre-hospital administration Cardiology 2002 98 141 7 12417813 10.1159/000066324 Nee P Gray AJ Martin MA Audit of thrombolysis initiated in an accident and emergency department Qual Health Care 1994 3 29 33 10136256 Arbaugh W Shankar N Wan J Your 802.11 Wireless Network has No Clothes Access Client/Server Software Bailey B Scott JM Brown LH Public safety answering point readiness for wireless E-911 in New York State Prehosp Emerg Care 2003 7 199 203 12710778 Chin T Is wireless technology ready to roll? Health Data Manag 1998 6 78 82 84-6, 88-9 10179886 Duncan R Shabot M An enterprise web viewing system for clinical and administrative data Proc AMIA Symp 2000 1169 11080023 Duncan R Shabot MM Secure remote access to a clinical data repository using a wireless personal digital assistant (PDA) Proc AMIA Symp 2000 210 4 11079875 Jacobus P Secure and silent messaging – the wireless way Health Manag Technol 2000 21 18 11187249 Lippoff O Wireless invasion: health care's evolution to wireless connectivity J Med Pract Manage 2001 16 269 72 11345888 Nakamura M Yang Y Kubota S Shimizu H Miura Y Wasaki K Shidama Y Takizawa M Network system for alpine ambulance using long distance wireless LAN and CATV LAN Igaku Butsuri 2003 23 30 9 12832862 Jovanov E Raskovic D Price J Chapman J Moore A Krishnamurthy A Patient monitoring using personal area networks of wireless intelligent sensors Biomed Sci Instrum 2001 37 373 8 11347420 Orlov O Drozdov DV Doarn CR Merrell RC Wireless ECG monitoring by telephone Telemed J E Health 2001 7 33 8 11321707 10.1089/153056201300093877 Tan K Hinberg I Effects of a wireless local area network (LAN) system, a telemetry system, and electrosurgical devices on medical devices in a hospital environment Biomed Instrum Technol 2000 34 115 8 10820639 Gibby G Schwab WK Miller WC Evaluation of electrosurgical interference to low-power spread-spectrum local area net transceivers J Clin Monit 1997 13 345 8 9495285 10.1023/A:1007499527678 Hagihira S Takashina M Mori T Taenaka N Mashimo T Yoshiya I Infrared transmission of electronic information via LAN in the operating room J Clin Monit Comput 2000 16 171 5 12578100 10.1023/A:1009911208828 Intercepting Mobile Communications: The Insecurity of 802.11 Security of the WEP algorithm Wireless Local-Area Networking

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==== Front BMC Health Serv ResBMC Health Services Research1472-6963BioMed Central London 1472-6963-4-231534742610.1186/1472-6963-4-23Research ArticleImproving prescribing of antihypertensive and cholesterol-lowering drugs: a method for identifying and addressing barriers to change Fretheim Atle [email protected] Andrew D [email protected] Signe [email protected] Informed Choice Research Department, Norwegian Health Services Research Centre, P.O. Box 7004, St. Olavs plass, Oslo, Norway2004 3 9 2004 4 23 23 15 3 2004 3 9 2004 Copyright © 2004 Fretheim et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background We describe a simple approach we used to identify barriers and tailor an intervention to improve pharmacological management of hypertension and hypercholesterolaemia. We also report the results of a post hoc exercise and survey we carried out to evaluate our approach for identifying barriers and tailoring interventions. Methods We used structured reflection, searched for other relevant trials, surveyed general practitioners and talked with physicians during pilot testing of the intervention. The post hoc exercise was carried out as focus groups of international researchers in the field of quality improvement in health care. The post hoc survey was done by telephone interviews with physicians allocated to the experimental group of a randomised trial of our multifaceted intervention. Results A wide range of barriers was identified and several interventions were suggested through structured reflection. The survey led to some adjustments. Studying other trials and pilot testing did not lead to changes in the design of the intervention. Neither the post hoc focus groups nor the post hoc survey revealed important barriers or interventions that we had not considered or included in our tailored intervention. Conclusions A simple approach to identifying barriers to change appears to have been adequate and efficient. However, we do not know for certain what we would have gained by using more comprehensive methods and we do not know whether the resulting intervention would have been more effective if we had used other methods. The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial. ==== Body Background Much research has been carried out with the aim of influencing the performance of clinicians. The results have varied [1,2]. As with any human behaviour, clinical practice is difficult to change. Some strategies that have been evaluated, like passive dissemination of clinical practice guidelines, have had little or no effect on practice [3]. Others, like educational outreach visits ("academic detailing") and multifaceted interventions, may be more effective than passive interventions [1]. The reasons why clinical practice sometimes is not consistent with current best evidence varies across clinical problems and from one clinician to another. A logical consequence of this is to tailor quality improvement strategies to address specific barriers [4]. Several trials of tailored interventions have been conducted. The methods used for identifying barriers to change have varied and there is limited evidence of the relative usefulness of different approaches. However, the choice of method for identifying barriers has implications, particularly with regards to resources, since some methods are time consuming and demand the involvement of many individuals. This represents a practical and financial constraint. On the other hand, if such approaches lead to the identification of important barriers that otherwise would have been overlooked, they may be worth the effort. In this article we describe a simple approach we have used to identify barriers to changing professional practice. This was done as the first step in a process of developing an intervention to improve the pharmacological management of hypertension and hypercholesterolaemia [5]. The intervention focused on three specific recommendations in clinical practice guidelines for hypertension and hypercholesterolaemia [6-8] based on evidence of a gap between the recommendations and current practice in Norway: • Contrary to recommendations, physicians seem to rarely estimate the risk of cardiovascular disease before initiating treatment [9] • Sales of thiazides are low, despite these drugs being recommended as first-line medication [10] • Relatively few patients reach recommended treatment goals [11,12] We also report the results of a post hoc exercise and a survey we carried out to evaluate our approach to identifying barriers and interventions. Methods We developed the intervention through a process of identifying barriers to implementation of recommendations and measures specifically addressing these barriers ("tailoring"). The methods we used were structured reflection, searching for other relevant trials targeted at improving the management of hypertension or hypercholesterolaemia, conducting a survey among general practitioners and discussion with physicians during pilot testing of the intervention. Structured reflection The three authors reflected over possible barriers based in part on our own experience as physicians working in primary care in Norway. We used a worksheet to structure our reflection (see Additional file 1). The worksheet included factors that might act as barriers in the practice environment, the professional environment, and related to physicians' knowledge, skills and attitudes. One worksheet was completed for each targeted behaviour: increasing the use of cardiovascular risk assessment before initiating treatment for hypertension or hypercholesterolaemia, increasing the prescribing of thiazides for the treatment of uncomplicated hypertension, and increasing the proportion of patients on medication for hypertension and hypercholesterolaemia that reach recommended treatment goals. The worksheet was used to facilitate our group discussion of possible interventions to address the identified barriers. Our research group had recently completed a trial of a strategy for guidelines implementation when we were planning this study [13]. In that study the multifaceted intervention consisted of several passive components. Information and materials were distributed by mail and to a large degree we relied on the physicians themselves to make an effort at changing their practice. The observed changes in practice were small. In another trial we had found that the use of active sick leave for back patients was significantly increased through a proactive intervention compared to a passive one [14]. Based on these experiences our research group decided to test an active strategy in this study. Therefore we decided to use outreach visits ("academic detailing") prior to considering specific barriers. We considered systematic reviews of interventions to improve professional practice when we designed our strategy [1]. We searched the Cochrane Group of Effective Care and Organisation of Care trial register for trials of interventions targeted specifically at the management of hypertension or elevated cholesterol in general practice. Questionnaire to physicians We surveyed general practitioners about some of the interventions about which we were uncertain after our structured reflection. The details of the survey have been described elsewhere [9]. Briefly, 265 physicians who had participated in an earlier trial conducted by our research group [13] were asked to complete a questionnaire as part of the study-evaluation. We used that opportunity to seek answers to the following questions: 1. Do physicians assess cardiovascular risk before prescribing antihypertensive or cholesterol-lowering drugs? 2. If not, would physicians be more likely to do so it they received a fee for this? 3. Do physicians comply with current regulations limiting the reimbursement of cholesterol-lowering drugs? The last question was asked for two reasons. Firstly, we were considering making risk assessment a condition for reimbursement of the drugs. Secondly, the existing regulations were a possible barrier to adhering to our recommendations because they conflicted with these. Pilot testing During pilot testing of the intervention at two practices, which were selected for convenience, comments from physicians relevant to possible barriers were noted. We also informally evaluated each component of the intervention. Post hoc focus groups and structured reflection exercise After we had finished designing the intervention we had the opportunity of testing our method of structured reflection at a gathering of international researchers in the Research Based Education and Quality Improvement group (ReBEQI) , December 2003. Each participant was asked to complete a worksheet to identify barriers and possible interventions related to the low use of thiazides among general practitioners. They were randomly allocated to four different groups where they collaborated on completing the worksheet. They were also asked to grade the importance of each barrier or intervention as minor, moderate or major. We disregarded those rated as minor. We compared the results from the four groups with the barriers and interventions we had identified. Post hoc survey of physicians exposed to the intervention While conducting the randomised trial to test the effectiveness of our multifaceted intervention we carried out telephone interviews with physicians allocated to the experimental group. They were asked if they adhered to our recommendations and, if not, why. The responses where noted down during the interviews. Results Barriers and interventions Figure 1 illustrates the timeframe for the methods used to identify barriers and interventions. Tables 1, 2, 3 give an overview of the barriers and interventions that we identified for each clinical problem. Many of the barriers were related to a lack of knowledge and could be addressed through educational interventions. The use of educational outreach visits was logical since we had planned to use an active intervention, based on our previous experience, and since this type of intervention has consistently lead to improved professional behaviour in randomised trials [1]. Similarly, based on previous experience and the capabilities of the software we hade developed [13], we planned on using an electronic risk calculator, electronic prescriptions, patient information materials, and computerised reminders. The search (July 2001) of the EPOC trial register for randomised trials with the word "hypertension" in any field yielded 58 references. Most were excluded after reading the abstracts, leaving eight, for which the full text was reviewed [15-22]. This did not lead to any changes in our intervention strategy. A search for randomised trials with the word "cholesterol" yielded 13 references. The full text was reviewed for only one of these [23]. This also provided little further guidance for designing our intervention. The nine trials that we reviewed are summarised in table 4. The survey results did not indicate that a fee for estimating cardiovascular risk before initiating drug therapy would affect practice [9]. The survey results also indicated that physicians are largely not affected by conditions for drug reimbursement [9]. Moreover, there were no mechanisms in place to enforce such regulations. We did not identify additional barriers during pilot testing of the intervention with five physicians in two practices, but several of those already identified were confirmed, particularly barriers to prescribing thiazides. Based on our findings and an assessment of the feasibility and evidence of effectiveness for various interventions, we designed a multifaceted intervention. The elements of the intervention are described in table 5. We also considered a number of interventions that we excluded. For example: • We considered placing computers in waiting rooms so that patients could assess their cardiovascular risk before seeing the physician, but concluded this would be costly and difficult to implement. • We considered providing pre-printed prescriptions, but found this would not to be relevant because most physicians use computerised systems for prescribing. • We considered exposing conflicts of interest among clinical specialists who advocated using other first line drugs than thiazides, but elected not to do so. • We considered exposing techniques used in pharmaceutical advertisements, such as using relative risk reductions rather than absolute risk reductions [24], but concluded this would have at best a limited impact. Post hoc focus groups and structured reflection exercise Nineteen researchers were divided into four groups. All groups considered advocacy by drug companies to be a major barrier to change. Routines or habits were also included as an important barrier by all the groups, as well as lack of knowledge concerning the effectiveness of thiazides, their favourable adverse effects profile, and their low cost. All the groups also mentioned competing guidelines or diverging opinions as part of the problem. Three of the groups considered local or national opinion leaders as potential barriers to change. Patients' expectations or perceived expectations were also mentioned by three of the groups. The interventions recommended by the groups to address the identified barriers are presented in table 6. All the groups suggested the use of computerised reminders to address physicians' lack of knowledge or their habits and routines. All the groups also suggested some form of interactive education, mainly as a counter force to promotional activities by the pharmaceutical industry, and patient information was suggested by three of the groups. Two suggested training physicians to address patient expectations. Two groups suggested developing clinical guidelines and two suggested audit and feedback, but one group considered this to be of minor importance. Post hoc survey of physicians exposed to the intervention Among the 195 physicians exposed to the intervention, 149 (76%) were contacted during the trial period and agreed to answer our questions. No major additional barriers were identified. However, some physicians questioned whether adhering to the recommendations would represent a good use of resources, specifically the recommended treatment goals. Discussion Addressing barriers to change with tailored interventions makes sense and there is some empirical support for this [1]. It is unclear, however, what methods are the most useful for identifying barriers and interventions. Several qualitative methods can be used to identify barriers, such as interviews, focus groups and observation. These methods may be valuable, but they are relatively labour-intensive. We used a simpler approach to identifying barriers to change. Would the use of other methods have provided us with important additional information? Pilot testing and discussions with five physicians in two practices and interviews with 140 participating physicians did not indicate additional barriers. The post-hoc focus groups with international experts did not add much with regards to barriers and interventions. Several of these groups included "routines and habits" as a potential barrier, which was not explicitly mentioned among the barriers identified by the investigators. However, all interventions that were mentioned by more than one of the groups in the post-hoc focus group exercise were included in our multifaceted intervention. Our use of computerised reminders was based on the assumption that this would help to establish new routines, although we did not record routines and habits as a barrier when we developed the intervention. There are inherent weaknesses in our approach. One is that the investigators undertaking the structured reflection were few and we were prejudiced by our own experiences. The lack of patient involvement is another limitation, which possibly lead to an under-emphasis of patient-mediated interventions. A weakness with the group of international researchers who participated in the post-hoc focus groups is their lack of familiarity with the Norwegian context. A number of trials of tailored interventions have been conducted. The methods used to identify barriers to change have varied. Some investigators have simply hosted a meeting [25,26], others have used questionnaires [23], conducted focus-groups [27-30] or interviews [31-33], or both [34]. Others have used a combination of several qualitative methods [35-37]. Some investigators have used identification of barriers as an intervention in itself [19,38,39]. The methods that were used have been poorly described in most of these studies. Conclusions Our simple approach to identifying barriers to improving practice appears to have been effective in identifying all of the important barriers, and it was efficient. However, we do not know for certain what barriers other methods would have identified or whether the intervention could have been more effective, if we had used other methods. Further work to address these questions is planned, including direct comparison of alternative methods and evaluations of theory-based approaches . The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial. Competing interests None declared. Author contributions All the authors participated in the process of structured reflection and in conducting the survey of physicians. AF was responsible for reviewing the results from previous research and pilot testing of the intervention. AF drafted the article while SF and ADO contributed to critical revisions of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank Kari Håvelsrud for her contributions in carrying out the surveys among physicians, and the ReBEQI-group (Research Based Education and Quality Improvement) for participating in the post hoc exercise. We also thank Dr. E. Borna in Oslo, Norway, and the physicians at Sentrumsklinikken, Asker, Norway for piloting the intervention, and the 149 physicians who participated in the telephone interviews. This work was carried out as part of a project funded by the Norwegian Ministry of Health and the Norwegian Directorate for Health and Social Affairs. Figures and Tables Figure 1 Flow chart indicating time frame for methods used to identify barriers and interventions. * The trial period ended in December 2003, and data collection and analysis will be completed in September 2004. Table 1 Barriers to carrying out cardiovascular risk assessment, and possible interventions to address these Possible barriers Possible interventions Time-consuming procedure for the physician - Easy-to-use tools (e.g. risk calculator for computer) - The patients or an assistant could carry out the risk assessment - Offer economic compensation Physician has no risk assessment tool at hand - Provide risk assessment tool The patients are focused on single risk-factors, not the global picture - Patient-information Physicians are not used to risk estimation, not educated to do this - Educational outreach visit - Training Lack of knowledge among physicians of the relevance of global risk assessment - Information/education (outreach visit) Physicians have more trust in their own clinical judgement than tables or charts - Education (e.g. case discussions during outreach visit) Differences in opinion among physicians on the importance of treatment of hypertension and hypercholesterolaemia - Use opinion-leaders and convince clinicians of the high degree of consensus in clinical guidelines May be uncomfortable for physicians to discuss risk-factors with patients - Patient-information - Offer strategies on how to communicate such information with patients Table 2 Barriers to prescribing thiazides for the treatment of hypertension, and possible interventions to address these Possible barriers Possible interventions Physicians are neither familiar with the relevant brand-names nor to the use and follow-up of these drugs - Pre-printed prescriptions, also in electronic format - Patient information - Support for the clinical follow-up Few other clinicians use these drugs - Patient information - Active promotion of thiazides (through educational outreach visits) - Point out the consensus among guidelines that thiazides are a first-line drug Specialists may be prescribing other drugs - Identify opinion leaders that advocate the use of thiazides - Look into possible conflicts of interest Advocacy by pharmaceutical companies - Point attention to the importance of clinically relevant endpoints when studies are quoted (during educational outreach visits) - Review advertisements to identify the main lines of reasoning that are being used Physicians are worried about possible side-effects and lack of anti-hypertensive effect. - Educational outreach visits Thiazides considered old-fashioned - Argue that these drugs have been thoroughly tested over many years (during educational outreach visits) Table 3 Barriers to reaching recommended treatment goals and possible interventions to address them Possible barriers Possible interventions Physicians are not accountable to anyone - Feed-back on to what extent treatment goals are reached among his/her pool of patients (audit) Physicians are unsure of what treatment goal to use - Give clear treatment goals (during outreach visit) - Point at the relatively high degree of consensus among guidelines (during outreach visit) Reluctance and unclear strategy among physicians on how to deal with insufficient treatment - Support for decision-making if goal is not reached Physicians may be underestimating the consequence of under-treatment - Educational outreach visit Table 4 Studies targeting the management of hypertension and/or hypercholesterolaemia Study Objective Intervention Comment Bass 1986 [15] Improve the detection and management of hypertension Medical assistant oversaw screening, attended to education, compliance and follow-up We did not consider this to be a feasible intervention in our setting Aucott 1996 [19] Implement guidelines for cost-effective management of hypertension on medication use and cost, blood pressure control, and other resource use Intensive guideline-based education and supervision (identification of clinical champion, faculty education and development, assignment of PharmD, clinic-based education and precepting of clinicians, monthly feedback to practice) Most elements of this multifaceted intervention were already included in our own. The trial was conducted in a general internal medicine teaching clinic, which limits the relevance to our primary care setting Rossi 1997 [16] Alter prescribing habits for the treatment of hypertension Guideline reminders placed in the charts of patients Computerised reminders were already included as part of our multifaceted intervention Goldberg 1998 [17] Increase compliance with national guidelines for the primary care of hypertension (and depression) Academic detailing with or without continuous quality improvement (CQI) teams Academic detailing (outreach visit) was already included as part of our multifaceted intervention. The study-findings did not support the use of CQI teams Maclure 1998 [18] Increase understanding of the way in which dissemination of evidence changes medical practice Media stories, national warning letter, teleconference, small group workshops, and newsletters Our outreach visits were planned as interactive sessions, thus serving the same purpose as small group workshops or teleconferences. We did not believe that passive distribution of material would be useful Hetlevik 1998 [21] Implement clinical guidelines in the treatment of hypertension Computer based clinical decision support system, mailed feedback of current practice, invitation to seminar at conference Most interventions were already included in our multifaceted intervention. We did not believe that inviting to conference-seminar would be useful van der Weijden 1999 [23] Assess the feasibility and evaluation needs of a cholesterol guideline Group education, desktop supportive materials, feedback on performance, and face-to-face instruction on location Most interventions were already included in our multifaceted intervention Montgomery 2000 [20] Have an effect on absolute cardiovascular risk, blood pressure, and prescribing of cardiovascular drugs Computer based clinical decision support system plus cardiovascular risk chart; or cardiovascular risk chart alone Both interventions were already included in our multifaceted intervention Demakis 2000 [22] Improve resident physician compliance with standards of ambulatory care (including hypertension) Computerised reminder system Intervention was already included in our multifaceted intervention Table 5 The final multifaceted intervention Educational outreach visit - Presentation focusing on three main messages:  1. Relevance of risk estimation and how to do it, including strategies on how to communicate information about risk to patients.  2. Information on evidence in support of effect and the unjustified fear of adverse effects regarding thiazides, pointing at the consensus that exists among guidelines. Attention also directed to the importance of clinically relevant endpoints when studies are quoted.  3. Clear recommendations justified by referring to high degree of consensus among guidelines. - Guidelines handed out, directing attention to the authors (opinion leaders) Audit & feed-back at outreach visit - To what extent treatment goals are achieved. - Drug-choice profile on anti hypertensives - Level of risk among patients on treatment, compared to a sample (men 40–65 years) not on treatment Computerised reminders - Risk assessment - First-choice antihypertensive drugs - Treatment goals Risk assessment tools as charts and in electronic format Patient-information material - The relationship between single risk factors and global risk - Thiazides and beta-blockers. - Treatment goals Table 6 Interventions to address identified barriers (main results from post-hoc focus group and structured reflection exercise) Barrier: Marketing activities by pharmaceutical industry Importance of barrier* Interventions Importance of interventions* Group 1 3 - Competing approaches (educational materials; interactive educational workshops) 2 Group 2 3 - Outreach visits 3 Group 3 3 - Small group peer comparison 1–2 Group 4 3 - Continuing education system Not graded Barrier: Routines and habits Importance of barrier* Interventions Importance of interventions* Group 1 3 - Computerised reminders 3 - Audit and feedback 2 - Intention plus/trial of behaviour 2 Group 2 2 - Reminder 3 - Direct mail 3 Group 3 2/3 - Audit and feed back with peer comparison 1 Group 4 Not graded - Computerised reminders Not graded Barrier: Lack of knowledge Importance of barrier* Interventions Importance of interventions* Group 1 2 - Educational material/guidelines 2 - Interactive educational meetings 2 Group 2 2 - Information to patients 2 - Local quality circles 2 Group 3 Not graded - Financial incentives 3 - Reminders/Computerised Decisions Support Systems 2 Group 4 Not graded - Continuing medical education Not graded - Computerised reminders Barrier: Opinion leaders (or specialists) or competing guidelines Importance of barrier* Interventions Importance of interventions* Group 1 3 - Develop national guidelines 2 Group 2 3 - Use opinion leaders 3 Group 3 2 - Guidelines shared by primary and specialist physicians 1–2 Group 4 Not graded - Not explicitly addressed - Barrier: Patient expectations Importance of barrier* Interventions Importance of interventions* Group 1 2 - Patient materials 3 - Educational meetings for general practitioners 2 Group 2 Not mentioned - None - Group 3 Not graded - Skills programme training 2 Group 4 Not graded - Information leaflet to patients about options Not graded *1 = minor, 2 = moderate, 3 = major ==== Refs Grimshaw JM Shirran L Thomas R Mowatt G Fraser C Bero L Grilli R Harvey E Oxman A O'Brien MA Changing provider behavior: an overview of systematic reviews of interventions Med Care 2001 39 II2 45 11583120 10.1097/00005650-200108002-00002 Oxman AD Thomson MA Davis DA Haynes RB No magic bullets: a systematic review of 102 trials of interventions to improve professional practice CMAJ 1995 153 1423 1431 7585368 Freemantle N Harvey EL Wolf F Grimshaw JM Grilli R Bero LA Printed educational materials: effects on professional practice and health care outcomes The Cochrane Library 2001 Oxford: Update Software Oxman AD Flottorp S An overview of strategies to promote implementation of evidence base health care Evidence Based Practice 2001 2nd 101 119 Fretheim A Oxman A Treweek S Bjørndal A Rational Prescribing in Primary Care (RaPP-trial). A randomised trial of a tailored intervention to improve prescribing of antihypertensive and cholesterol-lowering drugs in general practice [ISRCTN48751230] BMC Health Services Research 2003 3 5 12657163 10.1186/1472-6963-3-5 Fretheim A Bjørndal A Oxman AD Dyrdal A Golding M Ose L Reikvam A Teisberg P Retningslinjer for medikamentell primærforebygging av hjerte- og karsykdom – hvem bør behandles? [Guidelines for pharmacological primary prevention of cardiovascular disease: who should be treated?] Tidsskr Nor Laegeforen 2002 122 2277 2281 12448268 Fretheim A Bjørndal A Oxman AD Dyrdal A Golding M Ose L Reikvam A Teisberg P Hvilke blodtrykkssenkende legemidler bør brukes for primærforebygging av hjerte- og karsykdommer? [Which antihypertensive drugs should be used in the primary prevention of cardiovascular disease?] Tidsskr Nor Laegeforen 2002 122 2283 2286 12448269 Fretheim A Bjørndal A Oxman AD Dyrdal A Golding M Ose L Reikvam A Teisberg P Hvilke kolesterolsenkende legemidler bør brukes for primærforebygging av hjerte- og karsykdommer? [Which cholesterol-lowering drugs should be used in the primary prevention of cardiovascular disease?] Tidsskr Nor Laegeforen 2002 122 2287 2288 12448270 Fretheim A Havelsrud K Flottorp S Oxman AD Påvirker takster og refusjonsregler praksis? [Do fees for service and regulations for reimbursement influence practice?] Tidsskr Nor Laegeforen 2003 795 796 12693118 Norwegian Institute of Public Health Drug Consumption in Norway 1997 – 2001 Oslo 2002 Svilaas A Risberg K Thoresen M Ose L Lipid treatment goals achieved in patients treated with statin drugs in Norwegian general practice Am J Cardiol 2000 86 1250 3 A6 11090801 10.1016/S0002-9149(00)01212-1 Westheim A Klemetsrud T Tretli S Stokke HP Olsen H Blood pressure levels in treated hypertensive patients in general practice in Norway Blood Press 2001 10 37 42 11332332 10.1080/080370501750183372 Flottorp S Oxman AD Havelsrud K Treweek S Herrin J A cluster randomised trial of tailored interventions to improve the management of urinary tract infections and sore throat BMJ 2002 325 367 370 12183309 10.1136/bmj.325.7360.367 Scheel IB Hagen KB Herrin J Oxman AD A randomized controlled trial of two strategies to implement active sick leave for patients with low back pain Spine 2002 27 561 566 11884902 10.1097/00007632-200203150-00002 Bass MJ McWhinney IR Donner A Do family physicians need medical assistants to detect and manage hypertension? CMAJ 1986 134 1247 1255 2871915 Rossi RA Every NR A computerized intervention to decrease the use of calcium channel blockers in hypertension J Gen Intern Med 1997 12 672 678 9383135 10.1046/j.1525-1497.1997.07140.x Goldberg HI Wagner EH Fihn SD Martin DP Horowitz CR Christensen DB Cheadle AD Diehr P Simon G A randomized controlled trial of CQI teams and academic detailing: can they alter compliance with guidelines? Jt Comm J Qual Improv 1998 24 130 142 9568553 Maclure M Dormuth C Naumann T McCormack J Rangno R Whiteside C Wright JM Influences of educational interventions and adverse news about calcium-channel blockers on first-line prescribing of antihypertensive drugs to elderly people in British Columbia Lancet 1998 352 943 948 9752816 10.1016/S0140-6736(97)11390-3 Aucott JN Pelecanos E Dombrowski R Fuehrer SM Laich J Aron DC Implementation of local guidelines for cost-effective management of hypertension. A trial of the firm system J Gen Intern Med 1996 11 139 146 8667090 Montgomery AA Fahey T Peters TJ MacIntosh C Sharp DJ Evaluation of computer based clinical decision support system and risk chart for management of hypertension in primary care: randomised controlled trial BMJ 2000 320 686 690 10710578 10.1136/bmj.320.7236.686 Hetlevik I Holmen J Kruger O Kristensen P Iversen H Implementing clinical guidelines in the treatment of hypertension in general practice Blood Press 1998 7 270 276 10321438 10.1080/080370598437114 Demakis JG Beauchamp C Cull WL Denwood R Eisen SA Lofgren R Nichol K Woolliscroft J Henderson WG Improving residents' compliance with standards of ambulatory care: results from the VA Cooperative Study on Computerized Reminders JAMA 2000 284 1411 1416 10989404 10.1001/jama.284.11.1411 van der Weijden T Grol RP Knottnerus JA Feasibility of a national cholesterol guideline in daily practice. A randomized controlled trial in 20 general practices Int J Qual Health Care 1999 11 131 137 10442843 10.1093/intqhc/11.2.131 Skolbekken JA Communicating the risk reduction achieved by cholesterol reducing drugs BMJ 1998 316 1956 1958 9641937 Soumerai SB McLaughlin TJ Gurwitz JH Guadagnoli E Hauptman PJ Borbas C Morris N McLaughlin B Gao X Willison DJ Asinger R Gobel F Effect of local medical opinion leaders on quality of care for acute myocardial infarction: a randomized controlled trial JAMA 1998 279 1358 1363 9582043 10.1001/jama.279.17.1358 A controlled trial to improve care for seriously ill hospitalized patients. The study to understand prognoses and preferences for outcomes and risks of treatments (SUPPORT). The SUPPORT Principal Investigators JAMA 1995 274 1591 1598 7474243 Flottorp S Oxman AD Tailoring interventions to improve the management of urinary tract infections and sore throat: a pragmatic study using qualitative methods BMC Health Services Research 2003 3 3 12622873 10.1186/1472-6963-3-3 Evans D Mellins R Lobach K Ramos-Bonoan C Pinkett-Heller M Wiesemann S Klein I Donahue C Burke D Levison M Levin B Zimmerman B Clark N Improving care for minority children with asthma: professional education in public health clinics Pediatrics 1997 99 157 164 9024439 10.1542/peds.99.2.157 Hux JE Melady MP DeBoer D Confidential prescriber feedback and education to improve antibiotic use in primary care: a controlled trial CMAJ 1999 161 388 392 10478162 Santoso B Suryawati S Prawaitasari JE Small group intervention vs formal seminar for improving appropriate drug use Soc Sci Med 1996 42 1163 1168 8737434 10.1016/0277-9536(95)00390-8 Avorn J Soumerai SB Everitt DE Ross-Degnan D Beers MH Sherman D Salem-Schatz SR Fields D A randomized trial of a program to reduce the use of psychoactive drugs in nursing homes N Engl J Med 1992 327 168 173 1608408 Majumdar SR Soumerai SB Lee M Ross-Degnan D Designing an intervention to improve the management of Helicobacter pylori infection Jt Comm J Qual Improv 2001 27 405 414 11480202 Avorn J Soumerai SB Improving drug-therapy decisions through educational outreach. A randomized controlled trial of academically based "detailing" N Engl J Med 1983 308 1457 1463 6406886 Leviton LC Baker S Hassol A Goldenberg RL An exploration of opinion and practice patterns affecting low use of antenatal corticosteroids Am J Obstet Gynecol 1995 173 312 316 7631711 10.1016/0002-9378(95)90220-1 Ross-Degnan D Soumerai SB Goel PK Bates J Makhulo J Dondi N Sutoto Adi D Ferraz-Tabor L Hogan R The impact of face-to-face educational outreach on diarrhoea treatment in pharmacies Health Policy Plan 1996 11 308 318 10160376 Forsetlund L Bjorndal A Identifying barriers to the use of research faced by public health physicians in Norway and developing an intervention to reduce them J Health Serv Res Policy 2002 7 10 18 11822256 10.1258/1355819021927629 Scheel IB Hagen KB Oxman AD Active sick leave for patients with back pain: all the players onside, but still no action Spine 2002 27 654 659 11884914 10.1097/00007632-200203150-00016 Baker R Reddish S Robertson N Hearnshaw H Jones B Randomised controlled trial of tailored strategies to implement guidelines for the management of patients with depression in general practice Br J Gen Pract 2001 51 737 741 11593835 Cranney M Barton S Walley T Addressing barriers to change: an RCT of practice-based education to improve the management of hypertension in the elderly Br J Gen Pract 1999 49 522 526 10621984

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==== Front Filaria JFilaria Journal1475-2883BioMed Central London 1475-2883-3-81530789210.1186/1475-2883-3-8ResearchComparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis Noordin Rahmah [email protected] Sitti [email protected] Andarias [email protected] Lim Boon [email protected] Maria [email protected] Erliyani [email protected] Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Penang, Malaysia2 Dept. of Parasitology, Hasanuddin University, Jalan Perintis Kemerdekaan KM 10 Tamalanrea 90245, Makassar, Indonesia3 Department of Parasitology, Leiden University Medical Centre, P.O Box 9600, 2300 RC, Leiden, The Netherlands2004 12 8 2004 3 8 8 19 2 2004 12 8 2004 Copyright © 2004 Noordin et al; licensee BioMed Central Ltd.2004Noordin et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. Methods Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid™]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. Results There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. Conclusions Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study. ==== Body Background Lymphatic filariasis affects approximately 120 million people worldwide. Ten percent of these infections are attributed to Brugia malayi and Brugia timori [1]. Thick blood smear examination is the routine parasitological method used for diagnosis and prevalence studies in the Brugia endemic countries of Malaysia and Indonesia [2,3]. This diagnostic method depends on the detection of microfilariae in the peripheral blood, and due to the nocturnal periodicity of microfilaremia in these areas, requires nighttime collection and survey, which is often unpopular with the local population. Furthermore, this method is relatively insensitive [4] and difficult to perform accurately and with consistency in field situations. Conversely, serological diagnostic methods exhibit better sensitivity than detection of microfilaria by thick blood smear, allow the detection of amicrofilaraemic infections among "endemic normals" and afford daytime finger-prick blood sampling (thus overcoming the inconveniences associated with night blood sampling, thereby encouraging greater cooperation with the local population and facilitate field work) [5]. However, reports on antigen detection test for brugian filariasis have not demonstrated high levels of sensitivity [6,7]. Thus in the absence of a good antigen detection test for Brugia infection, anti-filarial IgG4 assay may be the next best alternative for detection of brugian filariasis [8]. Anti-filarial IgG4 levels have been demonstrated to be elevated in active filarial infection [9-13] and decline post-treatment [14-17]. Detection of anti-filarial IgG4 antibodies has also been used for epidemiological assessment of filariasis [13,18,19]. Studies assessing antibody prevalence of lymphatic filariasis have employed assays that use either soluble worm antigens or recombinant antigens [12,13,18-21]. These antigens may not bind to the same set of anti-filarial antibodies and probably display different cross-reactivities to antibodies against other infections. Thus differences in the antigens employed may be expected to affect the results of antibody prevalence studies. Therefore, the present study aimed to make direct comparison of two antigens i.e. soluble adult worm antigen (SWA) and a recombinant antigen (BmR1), in IgG4 assays on the same set of serum samples. Previously, an ELISA employing soluble adult worm antigen (SWA-ELISA) had been performed to determine prevalence of anti-filarial IgG4 antibodies on a set of serum samples from Indonesia. These samples were collected from: 1. A transmigrant population that migrated from a non-filarial endemic region to an area endemic for Brugian filariasis and; 2. Life-long residents of the Brugian endemic area [22]. In the present study a rapid test based on B. malayi recombinant antigen (BmR1 dipstick [Brugia Rapid™]) and detection of IgG4 antibodies were evaluated using the same set of serum samples. The BmR1 dipstick test has previously been shown to be highly specific and sensitive for the detection of brugian filariasis. In a study involving four international laboratories, the BmR1 dipstick was found to be 93% sensitive and 100% specific when tested with 535 serum samples from patients with various infections and healthy controls [8]. In another multicenter validation study, 97% sensitivity and 99% specificity were recorded when the BmR1 dipstick was tested with 753 serum samples [23]. The present study demonstrated that interpretations of some aspects of the seroepidemiology of filarial infection are affected by the kind of antigen employed in the assay. Thus this study, which utilized the same set of serum samples on assays using two kinds of antigens, highlights the role of the kind of antigen employed in the comparison of results of prevalence studies. Materials and methods Sera and study population The details on the sera and study population are as described previously [22]. Briefly, serum samples were collected cross-sectionally from a total of 247 transmigrants and 133 life-long residents (LLR) from Budong-budong, a district of Mamuju Regency in South-Sulawesi, Indonesia, which is endemic for nocturnal-periodic B. malayi [24,25]. The samples from the transmigrant population are valuable as they could help determine the pattern of acquisition of infection with increasing length of exposure to Brugian filariasis. The transmigrant population had traveled to their new homesteads in groups; they came from the same village or region in Bali or Lesser Sunda islands as part of the government-sponsored relocation programme. Each year a new settlement was founded close to the former one (between 10 and 20 kilometers) which accommodated groups of transmigrants from 2–3 different regions together with migrants from Polmas, an over-populated area in South Sulawesi, to promote integration of different tribes. Transmigrants were grouped together according to the year of arrival in the new settlement. A total of 6 transmigrant units, settled between several months and 6 years prior to the survey, were included in the study together with 2 villages of indigenous Sulawesians (LLR), which were situated closely to the transmigrant areas. Those aged ≤ 15 years were classified as children, while adults were classified as those aged 16 years and older. The mean age of children in transmigrant population and LLR population was 10.2 years and that of adults was 32.6 years. Soluble worm antigen (SWA) Adult B. malayi worms were purchased from TRS labs, Athens, Georgia, USA. Female worms were freeze dried, ground to powder, dissolved in phosphate buffered saline (PBS), homogenized and slowly stirred overnight at 4°C. The protein concentration was determined by 2,2'-biquinoline-4,4'-dicarboxylic acid disodium salt hydrate (BCA) method before storage at -20°C. BmR1 dipstick This BmR1 dipstick (Brugia Rapid™) was performed as described previously and according to the instructions of the manufacturer [16, Malaysian Bio Diagnostics Research Sdn. Bhd., Bangi, Selangor, Malaysia]. The BmR1 recombinant antigen was expressed from Bm17DIII DNA sequence, GenBank accession no. AF225296. Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female B. malayi demonstrated that the DNA sequence is present in all of the five kinds of libraries (Rahmah et al., unpublished data). Preliminary immunohistological studies suggest that the expressed antigen is found in the epithelial membranes of the adult female uterus (Rahmah et al., unpublished data). Statistical Analysis The results obtained with BmR1 dipstick were compared to the results obtained previously with SWA-ELISA. The similarities and differences in the results were analyzed by comparing proportion of related samples using McNemar test; and comparing proportions of unrelated samples by using Pearson Chi-square (if indicated Fischer exact test was used instead). Results and Discussion Figure 1 shows the antibody prevalence to B. malayi, as determined by BmR1 dipstick assay and SWA-ELISA, in the transmigrant population who had resided for various lengths of time in the endemic area and the antibody prevalence in the LLR population. The first detection of IgG4 antibody by the BmR1 dipstick was recorded at 3 years post-exposure, the antibody prevalence increased from 0% in the new arrivals (≤ 1 month and 2–4 months) to 7.4%, 11.1%, 39.1% and 42 % in populations exposed to the infection for 3, 4, 5 and 6 years respectively. Comparison of IgG4 assays using the two kinds of antigen demonstrated similarities in three areas. First, using both SWA-ELISA and BmR1 dipstick, the total prevalence of specific IgG4 in the transmigrant population was found to increase with increasing length of residence in the endemic area. Thus the BmR1 dipstick test confirmed the previously reported finding which demonstrated that the development of anti-filarial IgG4 correlated with the duration of exposure in previously unexposed population [22]. Second, after a period of 5–6 years of being exposed to the filaria infection, anti-filarial IgG4 prevalence by BmR1 dipstick in the transmigrant population (42%) was comparable (p = 0.763) to the antibody prevalence in the LLR population (39.8%); this finding was also previously reported with SWA-ELISA [22]. Thus the results with BmR1 dipstick is in agreement with the previous finding that approximately 5–6 years of exposure is required for the anti-filarial IgG4 in the transmigrant population to reach levels comparable to life-long residents. This is also reported to be the period needed for the detection of microfilaria in the peripheral blood [22]. Third, the overall prevalence of anti-filarial IgG4 was found to be higher in males than in females, by both BmR1 dipstick (p = 0.019) and by SWA-ELISA (p = 0.001). Figure 1 IgG4 antibody prevalence in transmigrant population who resided in B. malayi endemic areas for various lengths of time and in life-long residents (LLR), as determined by BmR1 dipstick and SWA-ELISA. Differences between the two kinds of antigen were also observed (Figure 1). First, except for year 5 transmigrants (p = 0.146), the percent antibody prevalence recorded in the transmigrant population and in the LLR population by BmR1 dipstick were significantly lower than that detected by the SWA-ELISA i.e. year 3, p = 0.00; year 4, p = 0.00; year 6, p = 0.041; LLR, p = 0.00. Second, using SWA-ELISA, anti-filarial IgG4 was first detected at 2–4 months post-exposure in the population of transmigrants, while using BmR1 dipstick the first detection of IgG4 antibody was recorded after three years of residence in the endemic area. Figure 2a and 2b shows the IgG4 antibody prevalence among children and adult populations as determined by BmR1 dipstick and SWA-ELISA respectively. There was no significant difference detected in antibody prevalence between adults and children in the transmigrant population and in the LLR population when BmR1 dipstick was used (year 3, p = 0.594; year 4, p = 0.066; year 5, p = 0.907; year 6, p = 0.061; LLR, p = 0.074). Using SWA-ELISA, except for early transmigrant settlers (2–4 months residents), the IgG4 prevalence in the transmigrant population was reported to be significantly higher in adults than in children [22]; however in the LLR population, antibody prevalence in children was not significantly higher than in adults (p = 0.316). Figure 2 IgG4 antibody prevalence in transmigrant adult and children populations who resided in B. malayi endemic areas for various lengths of time and in life-long residents (LLR). a (top) IgG4 antibody prevalence as determined by BmR1 dipstick assay. b (bottom) IgG4 antibody prevalence as determined by SWA-ELISA. (Note: Previously published in Parasitology 2001; 122, pg. 636 [Reproduced with permission]). Thus the BmR1 dipstick test demonstrated that the establishment of anti-filarial IgG4 in the children and adults of the transmigrant population occur at comparable rates; this is not in agreement with the previous finding, using SWA-ELISA, that demonstrated IgG4 was established more rapidly in the adult population than in children [22]. The above observations may be due to a mixture of filarial antigens in SWA, some of which may recognize antibodies produced by exposed but not infected individuals, and/or by individuals who have cleared the infection either due to treatment or spontaneous death of worms. Although IgG4 detection significantly increases the specificity of antibody assays in brugian filariasis [12] the specificity of parasite extract-based assays and recombinant antigen-based assays may not be the same. In a recent study in a non-filaria endemic area in Brazil, the presence of Strongyloides antibody responses was found to be associated with higher antifilarial IgG4 responses in assay that uses crude filaria extract as compared to assay that uses B. malayi Bm14 recombinant antigen [26]. The discrepancy between the results of SWA-ELISA and BmR1 dipstick may also be partly due to the greater sensitivity of the former as compared to the latter. Out of 120 (of 381) discrepant results, 111 were positive by SWA-ELISA but negative by BmR1 dipstick. Out of these 111 samples, 38 had low ELISA titers (cut-off value > 4.02 but < 4.5). If the stringency of the cut-off value of the SWA-ELISA is increased from 4.02 to 4.5, then the discrepancy can be considerably reduced to 88 (of 381), with 73 positive by SWA-ELISA but negative by BmR1 dipstick. On the other hand, since BmR1 dipstick test has been reported to be highly specific [8,23], it is unclear why there were eight individuals who were positive by BmR1 dipstick but negative by SWA-ELISA. Due to the problem of maintaining the antigenicity of the BmR1 antigen when shipped from Malaysia to Netherlands, the dipstick (immunochromatography) assay, which can be transported at room temperature, was employed to test the BmR1 antigen. However the difference in the assay formats is unlikely to be the reason for the lower overall antibody prevalence levels seen with the latter. This is because we have previously shown that ELISA using BmR1 was less sensitive (albeit equally specific) than BmR1 dipstick test [27] in detecting B. malayi infection. Thus in this study if the ELISA format had been used to determine prevalence of IgG4 antibodies to BmR1 instead of the dipstick format, the SWA-ELISA would still be detecting significantly more positives than the BmR1-ELISA. Differences between the two antigens were also observed in the pattern of IgG4 antibody prevalence in children (Figure 2a &2b). Using BmR1 dipstick, positivity of the test in children was first demonstrated at 3 years (2 of 35, 5.7%), followed by no positive child detected at year 4 (0 of 17), positive antibody prevalence at year 5 (3 of 8; 37.5%) which is significantly greater than at year 3 (p = 0.011). This is followed by a nonsignificant decrease (p = 0.589) in antibody prevalence by year 6 (6 of 22; 27.3%). IgG4 prevalence at year 6 and LLR (30 of 87; 34.5%) was also found to be not significantly different (p = 0.542). After year 4, the IgG4 prevalence in children seemed to achieve a stable level that was similar to the antibody prevalence in the LLR population children. In adults, infection was also initially detected at year 3 and there appeared to be a pattern of increasing antibody prevalence with increasing time of residence (9.1% at 3 years; 16.7% at year 4, 40% at year 5; 53.6 % at year 6). Furthermore, the detection rate at year 6 was not significantly different (p = 0.766) from that seen in the LLR population adults (50%). Thus using the BmR1 dipstick, the increasing total IgG4 prevalence with exposure to brugian filariasis was mostly due to the increasing positive cases in the adult population. It is tempting to speculate that the differences in the pattern observed in children and adults are due to the greater rate of aborted infections and/or spontaneous clearance of the infection in children than in adults. The overall lower worm burden in children, due to physiological differences, may enable higher rate of spontaneous clearance of infections in children than in adults. The difference in physiology between adults and children has been demonstrated by the greater number of natural killer cells, and T-and B-lymphocytes in children as compared to middle aged people [28]. In addition CD4/CD8 T-cells have also been reported to decline from a young age onwards [29,30]. However, since this is a cross-sectional study in which data for each period of residence were obtained from different groups of individuals, this hypothesis could not be confirmed. Conversely, results of the SWA-ELISA demonstrated initial antibody positivity in children at 2–4 months, followed by increasing antibody prevalence with time of residence. IgG4 prevalence at year 6 was found to be significantly lower than the antibody prevalence in children of the LLR population (p = 0.007). In the adult population, except for the earlier initial detection, a similar pattern was observed with the results of the BmR1 dipstick i.e. increasing rate of antibody prevalence in the transmigrants with exposure; and the IgG4 prevalence at year 6 is not significantly different (p = 0.430) than the antibody prevalence in the LLR adult population. Conclusions This study highlights that assays using both BmR1 and SWA antigens demonstrate an increasing prevalence of specific IgG4 antibodies in the transmigrant population with increasing length of residence in an area endemic for brugian filariasis, and, by six years residency that the antibody prevalence was similar to that observed in the LLR population. This study also documented three main differences in results derived from assays using two antigens i.e. 1. Earlier detection and higher rate of antibody prevalence by SWA-ELISA as compared to the results demonstrated by BmR1 dipstick; 2. Similar rate of acquisition of antibody prevalence in children and adults by BmR1 dipstick; whereas by SWA-ELISA adults were found to become antibody positive faster than in children; 3. By BmR1 dipstick the increasing total prevalence of IgG4 with exposure was primarily due to the adult population, whereas by SWA-ELISA this was attributed to both children and adult populations. This study demonstrates that some aspects of seroepidemiology of Brugia malayi infection may vary with the kind of antigen used in the assay. Thus comparison of results of different studies must take into account the kind of antigen employed, especially if one study uses native antigen and another uses recombinant antigen. It would be interesting to compare seroepidemiological data of IgG4 assays using two different recombinant antigens on the same population. Competing interests Rahmah Noordin is the inventor of the commercialized BmR1 dipstick test (Brugia Rapid™) Authors' contributions RN drafted the paper and supplied the BmR1 dipstick test; SW & AM supplied the sera and edited the paper; LBH performed most of the statistical analysis; ES and MY conceived the study and contributed significantly in editing of the paper: In addition ES performed the BmR1 dipstick test and some statistical analysis. All authors read and approved the final manuscript. Acknowledgements This study was funded by European Commission (EC) grant, No. ICA4-CT-2001-10081 and The Netherlands Foundation for the Advancement of Tropical Research (W93-266). ==== Refs World Health Organization Lymphatic Filariasis: Reasons for Hope 1998 3 5 WHO/CTD/97.4. Rev 1 Ministry of Health Malaysia Annual report 2003 Department of Health, Republic of Indonesia, Direktorat Jenderal PPM & PL Guideline for determination of the endemic area of filariasis (Pedoman penyakit kaki gajah), Jakarta 2002 McCarthy J Nutman TB Diagnosis of lymphatic filariasis infection In Lymphatic Filariasis 2000 United Kingdom: Imperial College Press 127 150 Ramzy RM Helmy H el-Lethy AS Kandil AM Ahmed ES Weil GJ Faris R Field evaluation of a rapid-format kit for the diagnosis of bancroftian filariasis in Egypt East Mediterr Health J 1999 5 880 887 10983526 Lalitha P Eswaran D Gnanasekar M Rao KV Narayanan RB Scott A Nutman T Kaliraj P Gnanasekar M Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1 Microbiol Immunol 2002 46 327 332 12139392 Au AC Denham DA Steward MW Draper CC Ismail MM Rao CK Mak JW Detection of circulating antigens and immune complexes in feline and human lymphatic filariasis Southeast Asian J Trop Med Public Health 1981 12 492 498 7046073 Rahmah N Shenoy RK Nutman TB Weiss N Gilmour K Maizels RM Yazdanbakhsh M Sartono E Multicenter laboratory evaluation of Brugia Rapid dipstick test for detection of brugian filariasis Trop Med Int Health 2001 8 895 900 14516300 10.1046/j.1365-3156.2003.01102.x Ottesen EA Skvaril LF Tripathy S Poindexter RW Hussain R Prominence of IgG4 in the IgG response to human filariasis J Immunol 1985 134 2707 2712 2579154 Kwan-Lim GE Forsyth KP Maizels RM Filarial-specific IgG4 responses correlates with active Wuchereria infection J Immunol 1990 145 4298 4305 2258618 Kurniawan A Yazdanbakhsh M van Ree RR Aalberse R Selkirk ME Partono F Maizels RM Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis J Immunol 1993 150 3941 3950 8473742 Rahmah N Anuar AK Ariff RH Zurainee MN A'shikin AN Fadzillah A Maimunah A Haq JA Use of antifilarial IgG4-ELISA to detect Brugia malayi infection in an endemic area of Malaysia Trop Med Int Health 1998 3 184 8 9593356 Haarbrink M Terhell A Abadi K van Beers S Asri M de Medeiros F Yazdanbakhsh M Anti-filarial IgG4 in men and women living in Brugia malayi endemic areas Trop Med Int Health 1999 4 93 97 10206262 10.1046/j.1365-3156.1999.00367.x Wamae CN Roberts JM Eberhard ML Lammie PJ Kinetics of circulating IgG4 after diethylcarbamazine and ivermectin treatment of bancrotian filariasis J Infect Dis 1992 165 1158 1160 1583340 Kurniawan A Atkinson R Sartono E Partono F Yazdanbakhsh M Maizels RM Differential decline in filarial specific IgG1, IgG4 and IgE antibodies in Brugia malayi infected patients after diethylcarbamazine therapy J Infect Dis 1995 172 1567 1572 7594718 McCarthy JS Guinea A Weil GJ Ottesen EA Clearance of circulating filarial antigen as a measure of the macrofilaricidal activity of diethylcarbamazine in Wuchereria bancrofti infection J Infect Dis 1995 172 521 526 7622896 Rahmah N Lim BH Khairul Anuar A Shenoy RK Kumaraswami V Lokman Hakim S Chotechuang P Kanjanopas K Ramachandran CP A recombinant antigen-based IgG4 ELISA for the specific and sensitive detection of Brugia malayi infection Trans R Soc Trop Med Hyg 2001 95 280 284 11490997 Rahmah N Lim BH Azian H Ramelah TS Rohana AR Short communication: use of a recombinant antigen-based ELISA to determine prevalence of brugian filariasis among Malaysian schoolchildren near Pasir Mas, Kelantan-Thailand border Trop Med Int Health 2003 8 158 163 12581442 10.1046/j.1365-3156.2003.01004.x Lim BH Rahmah N Afifi SA Ramli A Mehdi R Comparison of Brugia-Elisa and thick blood smear examination in a prevalence study of brugian filariasis in Setiu, Terengganu, Malaysia Med J Malaysia 2001 56 491 496 12014770 Weil GJ Ramzy RM El Setouhy M Kandil AM Ahmed ES Faris R A longitudinal study of Bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up Am J Trop Med Hyg 1999 61 53 58 10432056 Engelbrecht F Oettl T Herter U Link C Philipp D Edeghere H Kaliraj P Enwezor F Analysis of Wuchereria bancrofti infections in a village community in northern Nigeria: increased prevalence in individuals infected with Onchocerca volvulus Parasitol Int 2003 52 13 20 12543143 10.1016/S1383-5769(02)00045-4 Terhell AJ Haarbrink M Abadi KS Maizels RM Yazdanbakhsh M Sartono E Adults acquire filarial infection more rapidly than children: a study in Indonesian transmigrants Parasitology 2001 122 633 640 11444616 10.1017/S0031182001007855 Rahmah N Taniawati S Shenoy RK Lim BH Kumaraswami V Khairul Anuar A Lokman Hakim S Noor Hayati MI Chan BT Suharni M Ramachandran CP Specificity and sensitivity of a rapid dipstick test (Brugia Rapid) in the detection of Brugia malayi infection Trans R Soc Trop Med Hyg 2001 95 601 604 11816429 10.1016/S0035-9203(01)90091-4 Slee WV Onderzoek naar het voorkomen van filarial te Mamoedjoe Geneeskundig Tijdschrift voor Nederlands-Indie 1930 70 444 450 Partono F Oemijati S Hudojo Joesoef A Sajidiman H Putrali J Clarke MD Carney WP Cross JH Malayan filariasis in Central Sulawesi (Celebes), Indonesia Southeast Asian J Trop Med Public Health 1977 8 452 458 614706 Muck AE Pires ML Lammie PJ Influence of infection with non-filarial helminths on the specificity of serological assays for antifilarial immunoglobulin G4 Trans R Soc Trop Med Hyg 2003 97 88 90 12886811 10.1016/S0035-9203(03)90033-2 Rahmah N Shenoy RK Rohana AR Comparison of two IgG4 assay formats (Elisa and rapid dipstick test) for detection of brugian filariasis Southeast Asian J Trop Med Public Health 2003 34 768 770 15115085 Sansoni P Cossarizza A Brianti V Fagnoni F Snelli G Monti D Marcato A Passeri G Ortolani C Forti E Lymphocyte subsets and natural killer cell activity in healthy old people and centenarians Blood 1993 82 2767 2773 8219229 Aldhous MC Raab GM Doherty KV Mok JY Bird AG Froebel KS Age-related changes of memory, activation, and cytotoxic markers on CD4 and CD8 cells in children J Clin Immunol 1994 14 289 298 7814458 Osugi Y Hara J Kurahashi H Sakata N Inoue M Yumura-Yagi K Kawa-Ha K Okada S Tawa A Age-related changes in surface antigens on peripheral lymphocytes of healthy children Clin Exp Immunol 1995 100 543 548 7774067

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Filaria J. 2004 Aug 12; 3:8

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==== Front Ann Clin Microbiol AntimicrobAnnals of Clinical Microbiology and Antimicrobials1476-0711BioMed Central London 1476-0711-3-161535020510.1186/1476-0711-3-16Case ReportTuberculosis presenting as immune thrombocytopenic purpura Ozkalemkas Fahir [email protected] Ridvan [email protected] Atilla [email protected] Tulay [email protected] Vildan [email protected] Esra [email protected] Beril [email protected] Halis [email protected] Division of Hematology, Department of Internal Medicine, Uludag University School of Medicine, Uludag University Hospital, 16059, Bursa, Turkey2 Department of Chest and Tuberculosis, Uludag University School of Medicine, Uludag University Hospital, 16059, Bursa, Turkey3 Department of Microbiology and Infectious Diseases, Uludag University School of Medicine, Uludag University Hospital, 16059, Bursa, Turkey2004 6 9 2004 3 16 16 9 6 2004 6 9 2004 Copyright © 2004 Ozkalemkas et al; licensee BioMed Central Ltd.2004Ozkalemkas et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Although various hematologic abnormalities are seen in tuberculosis, immune thrombocytopenic purpura is a rare event. Case Presentation We report a case of a 29 year-old male who was presented with immune thrombocytopenia-induced hemoptysis, macroscopic hematuria and generalized petechiae. The patient was found to have clinical, microbiological and radiological evidence of active pulmonary tuberculosis. The immune thrombocytopenic purpura was successfully treated with anti-tuberculous drugs combined with corticosteroids and high dose immune globulin therapy. Conclusion Immune thrombocytopenic purpura can be one of the hematological manifestations of tuberculosis which has a global prevalence with increasing incidence secondary to HIV infection. Tuberculosisimmune thrombocytopenic purpuraimmune thrombocytopenia ==== Body Background During the past 2 decades, tuberculosis -both pulmonary and extrapulmonary- has re-emerged as a major health problem worldwide. Hematologic abnormalities have been described in association with mycobacterial infections for almost 100 years. Patients with both pulmonary and extrapulmonary tuberculosis (TB) may demonstrate peripheral blood abnormalities and findings may be minimal or profound [1,2]. A comprehensive review of the literature reveals a few case reports documenting tuberculosis as a cause of severe hematologic conditions such as hemophagocytic syndrome, thrombotic thrombocytopenic purpura and immune hemolytic anemia. Immune thrombocytopenic purpura (immune TP) associated with tuberculosis is exceedingly rare event. We report the case of immune TP associated with tuberculosis that was presented with severe hemorrhagic diathesis. Case Presentation A 29-year-old previously healthy immigrant male patient from Kazakhstan was admitted to hospital with new-onset severe hemoptysis, macroscopic hematuria and extensive cutaneous petechiae on lower extremities. He appeared ill and poorly nourished. The patient was oriented and well cooperated, and there was no previous history of hematologic or liver or another disease and recent medication. He presented with unexplained weight loss of 2 months duration along with intermittent fever, night sweats and cough. The physical examination revealed a blood pressure of 100/70 mm/Hg, pulse 100/min, a temperature 37.2°C, extensive cutaneous petechiae on lower extremities, hemorrhagic bulla on tongue and on mucosa of oral cavity, and amphoric soufflé on apex of right chest. No organomegaly or lymphadenomegaly or evidence of another disease such as chronic liver disease was detectable. The initial complete blood count revealed a white blood cell 25.1 × 109/l (58% neutrophils, 29% bants, 9% lymphocytes and 4% monocytes), hemoglobin 11.2 gr/dl, hematocrit 36%, MCV 84 fl, reticulocytes 1% and platelet count 7.6 × 109/l. Erythrocyte sedimentation rate was 110 mm/h. A peripheral smear was remarkable for a paucity of platelets. Coagulation profile [prothrombin time (PT), activated-partial thromboplastin time (aPTT), fibrin degradation products (FDP)] were normal. A bone marrow aspiration demonstrated hypercellularity of all cell lines with normal maturation of myeloid and erythroid precursors. Megakaryocytes were increased in number with normal morphology. On bone marrow aspiration hemophagocytosis was not observed. A chest X-ray (Figure 1) and computed tomography (CT) (Figure 2) demonstrated bilateral patchy infiltrates and walled cavities on left and right upper lobes. Acid-fast bacilli were strongly positive in sputum (Figure 3). Bone marrow aspirate and urine for acid-fast bacilli were negative. Sputum culture yielded mycobacterium tuberculosis complex. The following laboratory studies were normal or negative: biochemical tests (glucose, urea, creatinine, uric acid, sodium, potassium, calcium, chloride, phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, bilirubin, total protein and albumin), rheumatoid factor, anti-nuclear antibody, anti-platelet specific antibodies, Coomb's tests, HIV, hepatitis B and C virus, blood culture, bone marrow aspirate culture and abdominopelvic CT. No granuloma or hemophagocytosis was detected on bone marrow biopsy. The patient was started on rifampin 10 mg/kg/d, isoniazid 5 mg/kg/d, ethambutol 25 mg/kg/d, pyrazinamide 40 mg/kg/d, pyridoxine 75 mg/d and intravenous immune globulin (IVIg) 1 g/kg/d given for 2 days. On day 2 of hospitalization and treatment of anti-tuberculous therapy prednisolone 1 mg/kg/d was added. On day 8, platelet counts started to increase and on day 12 of the treatment it reached to 187 × 109/l level. Patient improved on day 10 and he did not have any complaints on day 14; at time of discharge. He received a total of 6 red blood cell (RBC) units throughout hospitalization. During his hospitalization findings of hemolysis or gastrointestinal bleeding and massive bleeding in another site except hematuria and hemoptysis were not established. A complete blood count at discharge demonstrated a WBC 17 × 109/l, Hb 9.6 g/dl, and platelet count 310 × 109/l (Table 1). Corticosteroids were discontinued on day 14 of therapy and the patient was discharged and recurrent thrombocytopenia was not established after withdrawal of corticosteroid therapy. Ninety days after discharge, the patient was well with a platelet count of 300 × 109/l (Table 1) and he had no side effect thought to be secondary to anti-tuberculous drugs. Figure 1 Anteroposterior chest radiograph showing cavitary lesions in both lungs Figure 2 CT showing cavitary lesions in both lungs Figure 3 Sputum showing positivity of acid-fast bacilli Table 1 Characteristics of patient Day Hb (g/dl) Hct (%) WBC (×109) Plt (×109) Treatment 1 11.2 36 25.1 7.6 Anti-tbc drugs + IVIg 2 8 25 23.1 9.6 Anti-tbc drugs + IVIg+Pred+1 RBC Susp 3 8.1 25 22.7 17.7 Anti-tbc drugs+ Pred+ 2 RBC Susp 4 10.3 33 20.6 15.1 Anti-tbc drugs+ Pred 5 8.6 27 24.5 12.3 Anti-tbc drugs+ Pred+ 1 RBC Susp 6 8.2 24 25.4 4.0 Anti-tbc drugs + Pred + 2 RBC Susp 7 10.1 29 20.6 4.7 Anti-tbc drugs+ Pred 8 10.5 32 17.7 20.2 Anti-tbc drugs+ Pred 9 9.9 31 15.8 36.6 Anti-tbc drugs+ Pred 10 10.3 33 18 59 Anti-tbc drugs+ Pred 11 9.4 28 14 100 Anti-tbc drugs+ Pred 12 9.7 28 18.1 187 Anti-tbc drugs+ Pred 13 9.1 26 15.2 324 Anti-tbc drugs+ Pred 14 9.6 28 17 310 Anti-tbc drugs 45 12.3 37 15.5 304 Anti-tbc drugs 90 14 43 11 300 Anti-tbc drugs Abbreviations: Anti-tbc drugs: anti-tuberculous drugs, Pred: Prednisolone, Susp: Suspension Discussion Tuberculosis differs from many other infectious maladies in having particular social and geographic distributions. The disease was under control in developed nations and getting under control in developing nations, until the emergence of HIV infection and the advent of multidrug resistant strains of mycobacteria [3-5]. Various hematologic abnormalities such as anemia, leukocytosis, monocytosis, lymphopenia, leucopenia, thrombocytopenia, thrombocytosis, leukemoid reactions and pancytopenia have been seen in tuberculosis [1,2], but severe thrombocytopenia and presenting of tuberculosis as immune thrombocytopenic purpura is extremely rare and there are a few reports about tuberculosis induced immune thrombocytopenic purpura published in the world literature [6-10]. The case that we report could be confused by coincidental presentation of adult idiopathic thrombocytopenic purpura and tuberculosis, by drug-induced thrombocytopenia, thrombotic thrombocytopenic purpura (TTP)-hemolytic uremic syndrome (HUS), hemophagocytic syndrome and disseminated intravascular coagulation (DIC) associated with TB. Idiopathic thrombocytopenic purpura (ITP, also known as primary immune thrombocytopenic purpura) is an acquired disease of children and adults defined as isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia. Adult ITP typically has an insidious onset with long-lasting histories of purpura (thrombocytopenia for >6 months) and spontaneous remission is uncommon and is likely to be incomplete [11-13]. Steroids are the conventional first line therapy for adult ITP. Platelets counts increase within one week in responding patients and usually reach peak values by two to four weeks. However, in most patients, thrombocytopenia recurs when steroids are tapered or discontinued. Also in adult, IVIg is used when clinical situations require a transient increase of the platelet count and a typical response is an increase in platelet count several days after the infusions are initiated and return to the pretreatment level within several weeks [11,13]. In our case, we excluded the adult ITP not only by basing on standard criteria [12], but with response to steroids and IVIg therapy since thrombocytopenia did not recur after withdrawal of prednisone and IVIg therapy. Also we excluded other causes of thrombocytopenia such as hemophagocytic syndrome, TTP, combined autoimmune cytopenias with history, clinical and laboratory findings, and examination of bone marrow aspiration and biopsy that were described in case presentation. Several factors are known to cause bleeding in association with infections, of which thrombocytopenia is the most common. The etiology of thrombocytopenia in most cases appears to be increased destruction of platelets such as due to DIC or septicemia without evidence of DIC or platelet adherence to damaged vascular surfaces or direct platelet toxicity caused by the microorganism or involvement of bone marrow. Adult acute immune thrombocytopenic purpura is defined as a bleeding disorder in otherwise healthy person caused by transient destruction of platelets. Although the most important therapy for infection-related thrombocytopenia is that directed at the underlying infection, treatment decisions for immune thrombocytopenic purpura remain controversial and may include single or combination therapy with corticosteroids, intravenous immunoglobulin (IVIg) according to degree of thrombocytopenia or hemorrhage [11,14]. The case that we reported was presented with symptoms of phthisis lasting for more than 2 months and severe hemoptysis, macroscopic hematuria and extensive cutaneous bleeding findings lasting for one week. Based on the clinical, radiological (X-ray and CT of chest) findings, demonstration of positivity of acid-fast bacilli in sputum and with exclusion of other causes of thrombocytopenia, immune thrombocytopenic purpura due to pulmonary tuberculosis was diagnosed. We applied anti-tuberculous therapy combined with corticosteroids and IVIg because of severe thrombocytopenia and severe hemorrhagic diathesis. Clinically, steroids are known to ameliorate the purpuric bleeding in patients before the platelet count actually increases. The early effect is due to decrease of vascular permeability. The effect of the steroids in the thrombocytopenia is probably complex and it is late effect. The mechanism of action of IVIg is unclear, but studies suggest blockage of the Fc receptors of the reticuloendothelial cells and suppression of antibody production and binding which may be a result of anti-idiotype antibodies that bind antiplatelet antibodies and modulate the immune response [14]. In our case, corticosteroids were discontinued on day 14 of therapy and the patient was discharged and recurrent thrombocytopenia was not established after withdrawal of corticosteroid therapy. These observations suggest that tuberculosis is the cause of thrombocytopenia in our patient. He received a total of 6 RBC units throughout hospitalization. He did not have findings of Coomb's positive or negative hemolytic anemia or microangiopathic hemolytic anemia, hemophagocytic syndrome, gastrointestinal bleeding and massive bleeding to thoracic cavity. But he had severe hemoptysis and macroscopic hematuria. Ninety days after discharge, the patient was in well health with a platelet count of 300 × 109/l and he had no side effect thought to be secondary to anti-tuberculous drugs [15,16]. The patient is still in our follow-up without relapsing of thrombocytopenia. In conclusion, since the incidence of tuberculosis is currently increasing in worldwide countries and it may present with different hematologic manifestations, in case of immune thrombocytopenic purpura tuberculosis should also be recalled. Finally, further studies are needed in order to fully characterize the pathophysiology and immunological abnormalities in tuberculosis-related immune thrombocytopenic purpura. ==== Refs Goldenberg AS Rom WN, Garay S Hematologic abnormalities and mycobacterial infections Tuberculosis 1996 New York, Litlle, Brown and Comp 645 655 Mert A Bilir M Tabak F Ozaras R Ozturk R Senturk H Aki H Seyhan N Karayel T Aktuglu Y Miliary tuberculosis: clinical manifestations, diagnosis and outcome in 38 adults Respirology 2001 6 217 224 11555380 10.1046/j.1440-1843.2001.00328.x Enarson DA Murray JF Rom WN, Garay S Global epidemiology of tuberculosis Tuberculosis 1996 New York, Litlle, Brown and Comp 57 76 Aaron L Saadoun D Calatroni I Launay O Memain N Vincent V Marchal G Dupont B Bouchaud O Valeyre D Lortholay O Tuberculosis in HIV-infected patients: a comprehensive review Clin Microbiol Infect 2004 388 398 15113314 10.1111/j.1469-0691.2004.00758.x Paolo WF JrNosanchuk JD Tuberculosis in New York city: recent lessons and a look ahead Lancet Infect Dis 2004 4 287 293 15120345 10.1016/S1473-3099(04)01004-7 Ghobrial MW Albornoz MA Immune thrombocytopenia: a rare presenting manifestation of tuberculosis Am J Hematol 2001 67 139 143 11343388 10.1002/ajh.1093 Spedini P Tuberculosis presenting as immune thrombocytopenic purpura Haematologica 2002 87 ELT09 11836186 Madkaikar M Ghosh K Jijina F Gupta M Rajpurkar M Mohanty D Tuberculosis and immune thrombocytopenia Haematologica 2002 87 ELT38 12161383 Tongyoo S Vilaichone W Sukpanichnant S Auewarakul C Chaiprasert A Ratanarat R Permpikul C Thrombocytopenic purpura associated with military tuberculosis J Med Assoc Thai 2003 86 976 980 14650711 Bakhshi S Kabra M Iyer VK Arya LS Thrombocytopenic purpura as a presenting manifestation of tubercular lymphadenitis Indian J Pediatr 2003 70 993 994 14719790 Levine SP Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader B Thrombocytopenia caused by immunologic platelet destruction Wintrobe's Clinical Hematology 2004 Philadelphia, Lippincott Williams & Wilkins 1535 1554 Ali R Özkalemkas F Özçelik T Özkocaman V Ozan Ü Kimya Y Köksal N Bülbül-Baškan E Develioğlu O Tüfekçi M Tunalý A Idiopathic thrombocytopenic purpura in pregnancy: a single institutional experience with maternal and neonatal outcomes Ann Hematol 2003 82 348 352 12734677 10.1007/s00277-003-0665-6 George JN Rizvi MA Beutler E, Lichtman MA, Coller BS, Kipps TJ, Seligsohn U Thrombocytopenia Williams Hematology 2001 New York, McGraw-Hill Co 1495 1540 Levine SP Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader B Miscellaneous causes of thrombocytopenia Wintrobe's Clinical Hematology 2004 Philadelphia, Lippincott Williams & Wilkins 1566 1572 Stork CM Hoffman RS Rom WN, Garay S Toxicology of antituberculosis drugs Tuberculosis 1996 New York, Litlle, Brown and Comp 829 841 Yee D Valiquette C Pelletier M Parisien I Rocher I Menzies D Incidence of serious side effects from first-line antituberculosis drugs among patients treated for active tuberculosis Am J Respir Crit Care Med 2003 167 1472 1477 12569078 10.1164/rccm.200206-626OC

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Ann Clin Microbiol Antimicrob. 2004 Sep 6; 3:16

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Ann Clin Microbiol Antimicrob

10.1186/1476-0711-3-16

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==== Front Nucl ReceptNuclear Receptor1478-1336BioMed Central London 1478-1336-2-61533312910.1186/1478-1336-2-6ResearchDiurnal difference in CAR mRNA expression Kanno Yuichiro [email protected] Satoshi [email protected] Takuya [email protected] Takayuki [email protected] Yoshio [email protected] Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan2004 28 8 2004 2 6 6 23 7 2004 28 8 2004 Copyright © 2004 Kanno et al; licensee BioMed Central Ltd.2004Kanno et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The constitutive androstane receptor (CAR, NR1I3) plays a key role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes. Results The expression of CAR mRNA throughout the circadian rhythm is reported for the first time in phase with the clock gene Bmal1 and in antiphase with the clock-controlled gene Rev-erbα mRNAs, with a peak at Zeitgeber time (ZT) 20 and a trough at ZT8, and a peak/trough ratio of 2.0. The diurnal difference in CAR mRNA expression might underlie the 1.7-fold difference in the magnitude of the PB-dependent induction of CYP2B1/2 mRNA. Conclusion The circadian oscillation of xenosensor gene CAR mRNA expression is partially responsible for chronopharmacokinetics and chronopharmacology in disease. ==== Body Background The superfamily of nuclear hormone receptor comprises a group of transcription factors that play significant roles in response to a number of biological regulators. In addition to the pregnane X receptor (PXR, NR1I2), the constitutive androstane receptor [1] (CAR, NR1I3) plays a role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes, such as cytochrome P450 (CYP) 2Bs, 2C19, 3As, multidrug resistance-associated protein 2 (MRP2), UDP-glucuronosyltransferase (UGT1A1), and 5-aminolevlinic acid synthase 1 (ALAS1) [2-8]. In response to xenobiotic PB, and other PB-like ligands such as 1,4-bis [2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP) in rodents [9] and 6-(4-Chlorpphenyl)imidazo [2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in humans [10], high doses of acetaminophen [11], and bilirubin [12], CAR forms a heterodimer with retinoid X receptor alpha (RXRα) and subsequently binds to the direct repeat (DR-4) motifs in such as the phenobarbital (PB)-responsive enhancer module (PBREM) in the far upstream promoter regions of mouse, rat and human CYP2B genes. In contrast, androstanol and androstenol were initially identified as inverse agonists [13] that reverse the constitutive transactivating potency of CAR. Recently, the mRNA expression of nuclear receptors, such as peroxisome proliferater-activated receptor alpha (PPARα), retinoic acid receptor (RAR)-related orphan receptor (ROR) and RER-ERBα, have been reported to show circadian rhythms in the liver [14-17]. Hepatic PPARα mRNA and protein levels follow a diurnal rhythm which parallels that of circulating corticosterone. In addition, REV-ERBα expression is regulated by a circadian positive feedback loop attributable to the function of BMAL1/CLOCK heterodimers, and is negatively controlled by circadian negative lobe PER/CRY heterodimers [18,19]. Circadian variations in the chronopharmacokinetics and chronopharmacology of various drugs such as theophylline and propranolol have been recently reported [20]. Furthermore, daily fluctuations in hepatic P450 monooxygenase activities responsible for the first phase of metabolism of various xenobiotics are well known. For example, Cyp2a4, Cyp2a5, CYP7, and CYP3A are among those that show circadian rhythmicities [21-23] that result from the preceding rhythmic oscillations of transcription factors including nuclear receptors. We previously determined the transcriptional start site of the rat Car gene (Kanno et al., 2003), resulting in the discovery of the putative REV-ERBα/ROR responsive element (RORE) at around -1.2 kb on the basis of published genomic sequence (Mazny et al., accession number AC099236). Thus, expression of the Car gene is expected to occur in antiphase to that of the Per1 gene and in phase with the Bmal1 gene. In the present study, the expression profile of the Car gene in rat liver was studied in comparison with those of the clock gene Bmal1, clock-controlled gene Rev-erbα and CAR-dependent PB-inducible CYP2B1/2 gene. Results Rat hepatic expression of nuclear receptor CAR mRNA follows a circadian rhythm Apart from the clock gene BMAL1 and clock-directed gene REV-ERBα, a time-dependent profile of CAR mRNA expression was observed for the rat liver. CAR mRNA levels oscillated during the day in phase with BMAL1 and in antiphase with REV-ERBα mRNAs, with a peak at ZT20 and a trough at ZT8, and a peak/trough ratio of 2.0 (Figs. 1, 2A). The CYP2B mRNA expression profile was resembled to the circadian oscillation of CAR mRNA but in a much more blunted manner (Fig. 2B). Figure 1 Diurnal variations in CAR mRNA in the rat liver. Animals were sacrificed every 4 hours at Zeitgeber times (ZT) 4, 8, 12, 16, 20 and 24/0. mRNA levels of CAR, CYP2B1/2, BMAL1, REV-ERBα and GAPDH were amplified by semi-quantitative RT-PCR. After oligo(dT)-primed cDNA was synthesized from rat liver total RNA, PCR was conducted with an initial enzyme activation step at 95°C for 5 min followed by divergent cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 60 sec; CAR (27 cycles), CYP2B1/2, REV-ERBα and BMAL1(30 cycles), and GAPDH (24 cycles). The reaction products were separated by agarose gel electrophoresis and stained with ethidium bromide. Figure 2 Diurnal difference in CAR and CYP2B mRNA levels in the rat liver. Animals were sacrificed at ZT8 and ZT20 (n = 3–4), and CAR (A) and CYP2B (B) mRNA levels were measured by semi-quantitative RT-PCR as described in the legend to Fig. 1. The results were normalized against those for GAPDH. The columns and bars represent the means ± SD with a significant difference at *: p < 0.01 Diurnal difference in the induction of CYP2B by phenobarbital Since CAR is associated with the induction of metabolic enzymes such as CYP2B, CYP3A, and UGT1A1, the circadian rhythmicity of CAR mRNA expression may be reflected in the diurnal-difference of PB-induction of CYP2B1/2 mRNA. Therefore, we investigated the time-dependent difference of the effect of PB-treatment on the induction of CYP2B1/2 mRNA. CYP2B1/2 mRNA expression was comparatively evaluated at ZT13 and ZT1 after 5-hours of PB treatment during ZT8 to ZT13 (the minimum zone of CAR mRNA expression) and ZT20 to ZT1 (during which the expression of CAR mRNA was maximal), respectively. Hepatic CYP2B1/2 mRNA was induced 2.2-fold over the control level in the rats treated with PB between ZT8 and ZT13 [daytime treatment]. In contrast, it was increased by 3.8-fold of the control level when the rats were treated from ZT20 to ZT1 [nighttime treatment] (Fig. 3). These data suggest that the diurnal-difference in CYP2B1/2-induction might be affected by the circadian rhythm of CAR mRNA expression. Figure 3 Diurnal difference of CYP2B induction. Animals were sacrificed at ZT13 and ZT25/1 5-hour after the injection of PB (gray columns) or veihcle (black columns) during ZT8-13 [Day] and ZT20-1 [Night], respectively. Oligo(dT)-primed cDNA was synthesized from rat liver total RNA from each animal, and CYP2B mRNA levels were measured by STBR Green real-time RT-PCR. The results were normalized against those of GAPDH. The columns and bars represent the means ± SD with significant differences compared to the individual controls at *, #: p < 0.05 Discussion We previously reported that the induction of rat CYP2B1/2 by PB is absent in the lung in contrast to the marked response in the liver due to the improper splicing of CAR mRNA during its maturation. [24,25]. The longitudinal expression of CAR mRNA along the gastrointestinal tract increases from the duodenum to the terminal jejunum and then decreases toward the distal ileum while only marginal expression can be observed in the stomach and colon, implying a role for endogenous ligands such as bilirubin glucuronides secreted in the duodenum [26]. A single transcriptional start site was determined by comparison between the full-length mRNA and genomic sequences. In the present study, we investigated whether the expression of hepatic CAR mRNA shows circadian rhythmicity, because clock-controlled regulation is expected due to the presence of putative RORE in the promoter region and electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the RORE probe using nuclear proteins (data not shown). The CAR mRNA level was found oscillation daily with a peak at ZT20 and a nadir at ZT8. In contrast, BMAL1 mRNA peaked at ZT24/0 and hit the bottom at ZT12 with a 4-hours retardation, and REV-ERBα mRNA showed a peak at ZT8 and a trough at ZT20 exactly in antiphase with CAR mRNA (Fig. 1). In contrast to the self-sustained central clock present in the brain, peripheral circadian clocks are retrained by humonal factors such as glucocorticoid hormones [27,28], as reflected in the diurnal rhythms observed for PPARα and REV-ERBα. Glucocorticoids are also responsible for the induction of human CAR mRNA and protein via a distal glucocorticoid response element in the 5'-franking region of the gene [29], and the same might be true for its rat counterpart, which was inducible by dexamethasone (data not shown). PPARα mRNA levels followed a similar diurnal rhythm to that of the plasma level of corticosterone, which is low in the morning (around ZT2), and increases in the afternoon to reach a peak 2–3 hours before the lights out (ZT9.5). Therefore, CAR mRNA oscillation might not be retrained by the physiological diurnal variation of glucocorticoids in rats. Recently, bilirubin was reported to be an endogenous activator of the CAR gene, which is in turn associated with the induction of bilirubin metabolising proteins, such as organic anion transporter SLC21A6, glutathione-S-transferase (GST), UGT1A1 and MRP2. Blood-bilirubin level reaches a minimum at the end of the light period and a maximum at the end of the dark period [30]. It is probable that blood bilirubin may contribute to the retraining of CAR expression to optimise bilirubin clearance. Hepatic CYP2B1/2 mRNA level was found to be synchronized with the CAR mRNA oscillation (Fig. 1). In the clock-controlled gene cascade or network, the circadian rhythm of CYP2B1/2 mRNA expression might be partially, if not fully, explained by the hepatic CAR level. Furukawa et al. showed that hepatic P450-dependent monooxygenase activities measured by the O-dealkylation of 7-alkoxycoumarin fluctuate daily in F344 rats with high values during the dark period [31]. In addition, these fluctuations are regulated by a central clock present in the suprachiasmatic nucleus [32]. Further, cholesterol 7-α hydroxylase (CYP7), coumarin 7-α hydroxylase (Cyp2a4) and coumarin 15-α hydroxylase (Cyp2a5) exhibit circadian rhythmicities. These enzymes are transcriptionally regulated by albumin D-site-binding protein (DBP), which is another primary clock-controlled gene expressed according to a robust daily rhythm in the SCN and several peripheral tissues. Besides DBP, REV-ERBα is transactivated by the binding of the BMAL1-CLOCK heterodimer to the E-box motif in its enhancer region [33], and is down-regulated by the clock gene PER-CRY heterodimer. Neuronal PAS domain protein 2 (NPAS2) is highly related in primary amino acid sequence to CLOCK, being able to dimerize with BMAL1 as in the case of CLOCK. Furthermore, BMAL1-NPAS2 heterodimer was found to transactivate the same target genes as those of BMAL1-CLOCK such as Per1, Per2, Cry1 and Rev-erbα. Recently, the transcription of Alas1 gene encoding for the aminolevulinate synthase 1 (Alas1) that is rate-limitting enzyme in a heme biosynthesis was reported to be controlled in the circadian clock mechanism. Although Alas1 is regulated transcriptionally by CAR-modulators having DR4 motifs in the promoter region as well as CYP2B1/2, BMAL1-NPAS2 and BMAL1-CLOCK heterodimers would be responsible for the daily physiological fluctuation in phase with Rev-erbα [34]. The circadian transcription of CAR and CYP2B1/2 is likely directly, indirectly or in combination dominated by these peripheral clocks and clock-controlled genes. The direct role of RevErb in the regulation of CAR will have to be established in further studies. For example ChIP analysis would be required to show diurnal occupancy of the putative RORE in the CAR promoter, and it has not yet been shown that Rev Erb α can modulate the transcription of the CAR promoter. We were also interested in whether the PB-dependent induction of CYP2B1/2 mRNA is affected by the diurnal rhythm of CAR. As shown in Fig. 3, PB-treatment at night [ZT20-1] was 1.7-fold more effective than treatment during the daytime [ZT8-13] in terms of the induction of CYP2B1/2 mRNA. Although the timing of the injection of PB and monitoring of CYP2B1/2 mRNA levels adopted in this work might not have been optimal, the results obtained suggested that the diurnal difference in the expression of xenosensor genes may underlie chronopharmacokinetics and chronopharmacology in a clinical setting. Conclusions Nuclear receptor CAR mRNA expression oscillates during the day with a peak at ZT20 and trough at ZT8 in antiphase with REV-ERBα, as expected due to the presence of putative ROREs in the promoter region. Since the magnitude of PB-induction of CYP2B1/2 mRNA showed at least a 1.7-fold difference during the day, the diurnal-difference of CYP2B-induction by PB might be controlled by the circadian rhythm of CAR mRNA expression. Methods Animals and treatments Eight week-old male Wistar rats (Clea) were kept under a 12-hours light-dark (LD12:12) cycle and provided food and water ad libitum. After more than 2 weeks of housing, the rats were killed at Zeitgeber times (ZT) 0, 4, 8, 12, 16, 20 and 24: ZT0 was lights-on and ZT12 is lights-out. For the PB-induction of CYP2B, the rats were i.p. injected with PB at ZT8 and ZT20 and sacrificed at ZT13 and ZT1, respectively. The livers were then dissected and used for the isolation of total RNA. RNA analysis by RT-PCR and Real-Time RT-PCR Total RNA was extracted from rat liver homogenate using an RNeasy Kit (QIAGEN, Hilden, Germany). After incubation at 65°C for 10 min, the extracts were quickly placed in an ice-cold water bath. Oligo-dT primed cDNA was synthesized from 1 μg of total RNA using RTG You-Prime First-Strand Beads (Amersham Biosciences, NJ), and left at room temperature for 1 min. Reverse transcription was then performed at 37°C for 1 hour to obtain cDNA. PCR was next performed in a total reaction mixture (25 μl) containing 1 μl each of RT-reaction mixture, Ex Taq DNA polymerase (Takara, Japan) and each of primer pair. cDNA was amplified for 24 (GAPDH), 27 (CAR) or 30 (BMAL1, REV-ERVα, CYP2B) cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min in a thermal cycler. The reaction products were separated by agarose gel electrophoresis and analyzed by a Flour Imager (Amersham Biosciences) after staining with ethidium bromide. Real-time PCR was carried out for the quantitation of each transcript in a reaction mixture consisting of 2 μl of the cDNA, 1 μl each pair of primers, 21 μl of water and 25 μl of iQ SYBER™ Green Supermix (BIO-RAD, CA). PCR was performed with an initial enzyme activation step at 95°C for 5 min followed by 50 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 45 sec in a real-time DNA thermal cycler (iCycler™, BIO-RAD). The following oligonucleotides were used as forward and reverse primers, respectively: 5'-ACCAGTTTGTGCAGTTCAGG-3' and 5'-CTTGAGAAGGGAGATCTGGT-3' for CAR, 5'-GAGTTCTTCTCTGGGTTGCTG-3' and 5'-ACTGTGGGTCATGGAGAGCTG-3' for CYP2B1/2, 5'-AACATGGCACTGAGCAGGTCTCC-3' and 5'-GGCATGTCCTATGAACATGTACC-3' for REV-ERBα, 5'-GCAAACTACAAGCCAACATTTCTAT-3' and 5'-CTTAACTTTGGCAATATCTTTTGGA-3' for BMAL1, and 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amplified cDNA was quantitated by the number of cycles (or cross point) at which the fluorescence signal was greater than a defined threshold during the logarithmic phase of amplification. The results were shown relatively to the control level after normalization to that of GAPDH. Competing interests None declared. Authors' contributions K.Y. conceived of the study, carried out all experiments and drafted the manuscript. S.O. and T.H. contributed to the experiment, and N.T. participated in the design of the study and its coordination. Y.I. participated in the design of the study and drafted the manuscript in collaboration with K.Y. All authors read and approved the final manuscript. ==== Refs Baes M Gulick T Choi HS Martinoli MG Simha D Moore DD A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements Mol Cell Biol 1994 14 1544 1552 8114692 Honkakoski P Moore R Washburn KA Negishi M Activation by diverse xenochemicals of the 51-base pair phenobarbital-responsive enhancer module in the CYP2B10 gene Mol Pharmacol 1998 53 597 601 9547348 Sueyoshi T Kawamoto T Zelko I Honkakoski P Negishi M The repressed nuclear receptor CAR responds to phenobarbital activating the human CYP2B6 gene J Biol Chem 1999 274 6043 6046 10037683 10.1074/jbc.274.10.6043 Chen Y Ferguson SS Negishi M Goldstein JA Identification of constitutive androstane receptor and glucocorticoid receptor binding sites in the CYP2C19 promoter Mol Pharmacol 2003 64 316 324 12869636 10.1124/mol.64.2.316 Goodwin B Hodgson E D'Costa DJ Robertson GR Liddle C Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor Mol Pharmacol 2002 62 359 365 12130689 10.1124/mol.62.2.359 Kast HR Goodwin B Tarr PT Jones SA Anisfeld AM Stoltz CM Tontonz P Kliewer S Willson TM Edwards PA Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor J Biol Chem 2002 277 2908 2915 11706036 10.1074/jbc.M109326200 Sugatani J Kojima H Ueda A Kakizaki S Yoshinari K Gong QH Owens IS Negishi M Sueyoshi T The phenobarbital response enhancer module in the human bilirubin UDP-gulcuronosyltransferase UGT1A1 gene and regulation by nuclear receptor CAR Hepatology 2001 33 1232 1238 11343253 10.1053/jhep.2001.24172 Fraser DJ Zumsteg A Meyer UA Nuclear receptor constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene J Biol Chem 2003 278 39392 39401 12881517 10.1074/jbc.M306148200 Tzameli I Pissions P Schuetz EG Moore DD The xenobiotic compound 1,4-bis[2-(3,5-dichlorpyridyloxy)] benzene is an agonist ligand for nuclear receptor CAR Mol Cell Biol 2000 20 2951 2958 10757780 10.1128/MCB.20.9.2951-2958.2000 Maglich JM Parks DJ Moore LB Collins JL Goodwin B Billin AN Stoltz CA Kliewer SA Lambert MH Willson TM Moore JT Identification of novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes J Biol Chem 2003 278 17277 17283 12611900 10.1074/jbc.M300138200 Zhang J Huang W Chua SS Wei P Moore DD Modulation of acetaminophen-induced hepatotoxicity by xenobiotic receptor CAR Science 2002 298 422 424 12376703 10.1126/science.1073502 Huang W Zhang J Chua SS Qatanani M Han Y Granata R Moore DD Induction of bilirubin clearance by the constitutive androstane receptor (CAR) PNAS 2003 100 4156 4161 12644704 10.1073/pnas.0630614100 Forman BM Tzameli I Choi H-S Chen J Simha D Seol W Evans RM Moore DD Androstane metabolites bind to and deactivate the nuclear receptor CAR-β Nature 1998 395 612 615 9783588 10.1038/26996 Lemberger T Saladin R Vazques M Assimacopoulos F Staels B Desvergne B Wahli W Auwerx J Expression of the peroxisome proliferator-activated receptor α gene is stimulated by stress and follows a diurnal rhythm J Biol chem 1996 271 1764 1769 8576180 10.1074/jbc.271.3.1764 Ueda HR Chen W Adachi A Wakamatsu H Hayashi S Takasugi T Nagano M Nakahama K Suzuki Y Sugano S Iino M Shigeyoshi Y Hashimoto S A transcription factor response element for gene expression during circadian night Nature 2002 418 534 539 12152080 10.1038/nature00906 Andre E Conquet F Steimyr M Stratton SC Porciatti V Becker-Andre M Disruption of retinoid-related orphan receptor β changes circadian behavior, causes retinal degeneration and leads to vacillans phenotype mice EMBO J 1998 17 3867 3877 9670004 10.1093/emboj/17.14.3867 Torra IP Tsibulsky V Delaunay F Saladin R Laudet V Fruchart J-C Kosykh V Staels B Circadian and glucocorticoid regulation of Rev-erbα expression in liver Endocrinology 2000 141 3799 3806 11014236 10.1210/en.141.10.3799 Reppert SM Weaver DR Coordination of circadian timing in mammals Nature 2002 418 935 941 12198538 10.1038/nature00965 Nakajima Y Ikeda M Kimura T Honma S Ohmiya Y Honma K Bidirectional role of orphan nuclear receptor RORα in clock gene transcriptions demonstrated by a novel reporter assay system FEBS letters 2004 565 122 126 15135064 10.1016/j.febslet.2004.03.083 Bruguerolle B Chronopharmacokinetics. Current status Clin Pharmacokinet 1998 35 83 94 9739478 Lavery DJ Lopez-Molina L Margueron R Fleury-Olela F Conquet F Schibler U Bonfils C Circadian expression of the steroid 15α-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP Mol Cell Bio 1999 19 6488 6499 10490589 Noshiro M Nishimoto M Okuda K Rat liver cholesterol 7α-hydroxylase. Pretranslational regulation for circadian rhythm J Biol Chem 1990 265 10036 10041 1693613 Ohno M Yamaguchi I Ito T Saiki K Yamamoto I Azuma J Circadian variation of the urinary 6β-hydroxycortisol to cortisol radio that would reflect hepatic CYP3A activity Eur J Clin Pharmacol 2000 55 861 865 10805065 10.1007/s002280050708 Nakahama T Mizuno M Otuka Y Inouye Y Comparative study on the mode of action of chlorinated ethylenes on the expression of rat CYP forms J Health Sci 2001 47 278 287 10.1248/jhs.47.278 Kanno Y Aoki S Nakahama T Inouye Y Role of the defective splicing of mRNA in the lack of pulmonary expression of constitutively active receptor in rat J Health Sci 2003 49 541 546 10.1248/jhs.49.541 Kanno Y Moriyama T Ichikawa H Mizuno M Nakahama T Inouye Y Transcriptional determination of sexually dimorphic expression of nuclear receptor CAR in Wistar rats J Health Sci 2004 50 384 388 10.1248/jhs.50.384 Balsalobre A Brown SA Marcacci L Tronche F Kellendonk C Reichardt HM Schutz G Schiler U Resetting of circadian time in peripheral tissues by glucocorticoid signalling Science 2000 289 2344 2347 11009419 10.1126/science.289.5488.2344 Le Minh N Damiola F Tronche F Schutz G Schibler U Glucocorticoid hormones inhibit food-induced phase-shifting of peripheral circadian oscillators EMBO J 2001 20 7128 7136 11742989 10.1093/emboj/20.24.7128 Pascussi JM Busson-Le Coniat M Maurel P Vilarem MJ Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: identification of a distal glucocorticoid response element Mol Endocrinol 2003 17 42 55 12511605 10.1210/me.2002-0244 Minematsu S Watanabe M Tsuchiya N Watanabe M Amagaya S Diurnal variations in blood chemical items in Sprague-Dawley rats Exp Anim 1995 44 223 232 7556424 10.1538/expanim.44.223 Furukawa T Manabe S Ohashi Y Sharyo S Kimura K Mori Y Daily fluctuation of 7-alkoxycoumarin O-dealkylase activities in the liver of male F344 rats under ad libitum-feeding or fasting conditions Toxicol Lett 1999 108 11 16 10472805 10.1016/S0378-4274(99)00065-X Furukawa T Manabe S Watanabe T Sehata S Sharyo S Okada T Mori Y Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones Arch Toxicol 1999 73 367 372 10550478 10.1007/s002040050675 Ripperger JA Shearman LP Reppert SM Schibler U CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP Genes & Dev 2000 14 679 689 10733528 Krista K Cheng CL Reciprocal regulation of haem biosynthesis and the circadian clock in mammals Nature 2004 430 467 471 15269772 10.1038/nature02724

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==== Front Popul Health MetrPopulation Health Metrics1478-7954BioMed Central London 1478-7954-2-61527967510.1186/1478-7954-2-6ResearchEstimating age conditional probability of developing disease from surveillance data Fay Michael P [email protected] National Cancer Institute 6116 Executive Blvd., Suite 504 Bethesda, MD 20892-8317, USA2 (Current Address) National Institute of Allergy and Infectious Diseases, 6700 B Rockledge Drive MSC 7609, Bethesda, MD 20892-7609, USA2004 27 7 2004 2 6 6 15 7 2003 27 7 2004 Copyright © 2004 Fay; licensee BioMed Central Ltd.2004Fay; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Fay, Pfeiffer, Cronin, Le, and Feuer (Statistics in Medicine 2003; 22; 1837–1848) developed a formula to calculate the age-conditional probability of developing a disease for the first time (ACPDvD) for a hypothetical cohort. The novelty of the formula of Fay et al (2003) is that one need not know the rates of first incidence of disease per person-years alive and disease-free, but may input the rates of first incidence per person-years alive only. Similarly the formula uses rates of death from disease and death from other causes per person-years alive. The rates per person-years alive are much easier to estimate than per person-years alive and disease-free. Fay et al (2003) used simple piecewise constant models for all three rate functions which have constant rates within each age group. In this paper, we detail a method for estimating rate functions which does not have jumps at the beginning of age groupings, and need not be constant within age groupings. We call this method the mid-age group joinpoint (MAJ) model for the rates. The drawback of the MAJ model is that numerical integration must be used to estimate the resulting ACPDvD. To increase computational speed, we offer a piecewise approximation to the MAJ model, which we call the piecewise mid-age group joinpoint (PMAJ) model. The PMAJ model for the rates input into the formula for ACPDvD described in Fay et al (2003) is the current method used in the freely available DevCan software made available by the National Cancer Institute. ==== Body Background Fay, Pfeiffer, Cronin, Le, and Feuer [1] showed how to calculate the age-conditional probabilities of developing a disease (ACPDvD) from registry data. Throughout this paper we use "cancer" as our disease of interest, but the method applies to specific types of cancer as well as other diseases where information is collected by population based surveillance methods. Fay et al [1] provided a formula (see equation 1 below) to calculate ACPDvD after inputing the rate function by age of (1) first incidence of cancer per person-years alive, (2) death from cancer per person-years alive, and (3) death from other causes per person-years alive. Fay et al [1] used a simple piecewise constant model for the three rate functions, which have constant rates within each age group. Here we detail two more complicated models for the rates. The first model is a segmented regression model or joinpoint model for the rates, where the rate function is a series of linear functions that join at the mid-points of the age groups, and the rate function is constant before the first mid-point and after the last "mid-point" (because the last interval goes to infinity, the last "mid-point" is not really a mid-point at all, see below). We will call this model the MAJ (mid-age group joinpoint) model for the rates. In Figure 1 we show how both the piecewise constant model and the mid-age group joinpoint model apply to all invasive cancer incidence from the Surveillance Epidemiology and End Results (SEER) program of the U.S. National Cancer Institute in 1998–2000. Figure 1 uses the SEER 12 registries which cover about 14 percent of the U.S. population, covering 5 states (Connecticut, Hawaii, Iowa, New Mexico, Utah), 6 metropolitan areas (Atlanta, Detroit, Los Angeles, San Francisco-Oakland, San Jose-Monterey, Seattle-Puget Sound) and the Alaska Native Registry (see [2]). Similar graphs showing the MAJ model can be made for the other rates required in the calculations, death from cancer and death from other causes per person-years alive. Figure 1 SEER 12 all invasive cancer incidence rates, 1998–2000, all races, both sexes: Piecewise constant and mid-age joinpoint methods. Notice that the MAJ model gives a more smoothly changing and probably a better modeled rate. The only place where the MAJ model may not perform better than the piecewise constant model is at peaks or valleys, where there may be some bias. In Figure 1 we see that the smoothness of the MAJ appears to produce more plausible estimates for ages 0 through 85 and from ages 90 and above, and the only age group with a noteworthy bias problem is 85 to 90. Thus, for almost all of the age range the MAJ model is more plausible. A problem with the mid-age group joinpoint model is that it requires numeric integration for its calculation. The second model uses a series of piecewise constant values to approximate the mid-age group joinpoint model. We call this second model the PMAJ (piecewise mid-age group joinpoint) model. The PMAJ does not require numeric integration, so it is much faster than the MAJ model. The PMAJ model is a piecewise constant model that only differs from the piecewise constant model of Fay et al [1] in that the pieces are smaller and the corresponding values of the rates are motivated by the MAJ model. Starting with version 5.0, the freely available DevCan software [3] uses the PMAJ method. (There was a small calculation error in versions 5.0 and 5.1 that has been corrected in version 5.2). DevCan calculates ACPDvD or age conditional probability of dying from a disease for U.S. cancer data or for user supplied data. The outline of this paper is as follows. The review and overview section reviews the issues in estimating the age conditional probability of developing disease from surveillance data. This section includes a motivation for using this type of statistic to describe population data. The review and overview section additionally gives graphical descriptions of the MAJ and PMAJ methods. The paper is structured so that readers not interested in the details may skip the next two sections and the appendix, which give precise and notationally involved definitions of the MAJ estimators. The examples and discussion section gives examples of the estimator of ACPDvD using three different methods for estimating the rates, the simple piecewise constant method proposed in Fay et al [1], the MAJ method, and the PMAJ method. In supplimental material [see Additional file 1] we compare the PMAJ method with the method of Wun, et al [4], since the latter method was the method used in versions of the DevCan software before version 5.0. Review and overview Consider a surveillance program like the SEER program of the U.S. National Cancer Institute. This program attempts to count every incidence of cancer within the catchment area of the program. Because cancer is a disease in which the rates of the disease are highly dependent on age, in order to give interpretability to the counts within the SEER registries, we must somehow account for the age distribution in the popoulation. One simple and popular statistic is the age adjusted rate or directly standardized rate (DSR). In the SEER Cancer Statistics Review [2] DSRs are used to compare different cancer sites, trends on specific cancer sites over time, and rates by sex and race. The DSR is calculated by a simple weighted sum of the age specific rates for each 5 year age group, where the weights are proportional to the U.S. 2000 population. Thus, the DSR may be interpreted as the rates adjusted as if all the populations being compared had age distributions similar to the U.S. 2000 population. The DSRs are useful for gaining an overall picture of how the incidence and mortality of each cancer effects different populations (e.g., different races, SEER population at different times), while controling for the effect of differing age distributions between populations being compared. A disadvantage of the DSR is that it is hard to relate to an individual's risk. For example, Table I-4 of the SEER Cancer Statistics Review, 1975–2000 [2] states that the DSR for breast cancer for females for the years 1996–2000 is 135 per 100,000 person-years. The average American woman may wonder, how does that relate to my risk? Will I be likely to get breast cancer in my lifetime? If I am 40 years old now, what is my risk of getting breast cancer in the next 10 years given that I have survived to this old without getting it? These questions are the motivation for using the age conditional probability of developing disease (ACPDvD), and in order to estimate the ACPDvD for female breast cancer, we require information not only about the rate of female breast cancer but also about the rates of dying from female breast cancer and dying from other causes. The ACPDvD uses cross-sectional incidence and mortality rates to estimate the age-conditional probabilities of developing disease in a hypothetical cohort in which we assume the age specific rates do not change over time. This gives a personal interpretation to the cross-sectional data, allowing statements like the following: if the incidence and mortality rates remain at their present values (as observed in SEER 12, 1998–2000), then a female born today would have a 13.5% chance of developing breast cancer over her lifetime (see Table 2). We can also calculate ACPDvD over intervals. For example, a female who has reached 40 years old without developing breast cancer has a 1.5% chance of developing breast cancer by the time she is 50. Table 2 Age Conditional Probability of Developing Different Types of Invasive Cancers (in Percent) from SEER 12, 1998–2000 Start Age End Age Model All Invasive (Both Sexes) Prostat(Male) Breast (Female) Acute Lymphocytic Leukemia (Both Sexes) 0 20 Piecewise const 0.3158 0.0009 0.0015 0.0669 PMAJ, interval = .5 0.3260 0.0011 0.0021 0.0633 MAJ 0.3260 0.0011 0.0021 0.0633 0 50 Piecewise const 4.0690 0.2002 1.9188 0.0837 PMAJ, interval = .5 4.1657 0.2550 1.9492 0.0808 MAJ 4.1657 0.2550 1.9492 0.0808 40 50 Piecewise const 2.5260 0.2032 1.5131 0.0053 PMAJ, interval = .5 2.5976 0.2579 1.5169 0.0055 MAJ 2.5975 0.2579 1.5169 0.0055 0 Inf Piecewise const 42.0876 17.4952 13.6471 0.1154 PMAJ, interval = .5 41.7547 17.3375 13.5477 0.1121 MAJ 41.7574 17.3389 13.5485 0.1121 60 61 Piecewise const 1.2340 0.5989 0.3822 0.0009 PMAJ, interval = .5 1.0852 0.4946 0.3627 0.0009 MAJ 1.0852 0.4946 0.3627 0.0009 64 65 Piecewise const 1.2758 0.6131 0.3872 0.0009 PMAJ, interval = .5 1.4453 0.7440 0.4045 0.0010 MAJ 1.4453 0.7440 0.4045 0.0010 60 65 Piecewise const 6.0331 2.9128 1.8777 0.0042 PMAJ, interval = .5 6.0622 2.9492 1.8758 0.0044 MAJ 6.0622 2.9492 1.8759 0.0044 Calculation of the ACPDvD is somewhat complicated, and we describe the complications in relation to the simple DSRs. Consider first the age specific incidence rates which are used to calculate the DSRs. These rates simply count the number of incident cases of a particular disease (e.g., female breast cancer) within each age group and divide by the total number of person-years estimated by the population. For counts of a single year, the person-years are estimated by the mid-year population of the catchment area (for sex-specific cancers like prostate cancer or female breast cancer, we only use the population of the appropriate sex). Note that the incident cases may include individuals who have previously been diagnosed with the cancer and have developed a new primary cancer. For the ACPDvD for any specific disease we would like the rate of first incidence per person-years alive and disease-free. Thus, there are two difficulties, (1) the usual age specific incidence rates include persons with multiple primary cancers, and (2) the denominators include persons who have previously been diagnosed. Merrill and Feuer [5] discuss both difficulties and adjust for them creating risk-adjusted cancer incidence rates. Merrill and Feuer [5] study the effect of these adjustments for several cancer sites. To handle the first difficulty, (similar to [5]) we can remove cases where we have a record of a previous diagnosis of that particular type of cancer. Because the registries in SEER were not all begun at the same time, to avoid bias the DevCan program only searches the records for previous cancers back until the year when the last registry was added. This year is denoted the follow-back year. (If the disease of interest is any malignant cancer, then the difficulty is handled differently. Although at each cancer record we do not record what specific types of cancers were previously diagnosed for the person, we do know whether any tumors were previously diagnosed. Thus, if the disease of interest is any malignant cancer and if the record states there was a previously diagnosed tumor, then we assume that the previously diagnosed tumor was malignant, and do not count that case as a first incidence.) To handle the second difficulty, the additional person-years in the denominator, Merrill and Feuer [5] adjust the denominator by multiplying the age-specific population by 1 minus an estimate of the prevalence of the disease in the population. Merrill and Feuer [5] also estimate the prevalence of medical procedures which remove individuals from the at-risk population, such as hysterectomy which removes the risk of uterine cancers. In calculating the ACPDvD we use only first incident of the disease of interest as in [5], but we correct for the denominators in a different way using an assumption and some mathematics from the theory of competing risks. This second correction is detailed with precise mathematical notation in Fay et al [1]; here we give more heuristic arguments. In the following let the disease of interest be "cancer". The ACPDvD between ages x and y, given alive and cancer-free at age x, may be written as the fraction, To calculate the numerator, we integrate over the probability that the first cancer occurred at exactly age a. In math notation this probability is where fc(a) is a probability function representing the probability that the first cancer occurred at exactly age a. One key result described in Fay et al [1] is that fc(a) can be written as the product of two functions, λc(a) = the probability that the first cancer occurred at exactly age a, given the individual is alive just before age a, and Sa(a-) = the probability that the individual is alive just before age a. The function λc(a) is known as a cause-specific hazard function, and it is estimated by some function of the age-specific rates, such as the piecewise constant model of Fay et al [1] or the MAJ model introduced in this paper (see Figure 1). Using standard results for continuous survival data, we can write Sa(a-) as where λa(u) ( = the probability that the individual died at age u, given the individual is alive just before age u) is the usual hazard function. We estimate λa(u) using some function of the age-specific rates. Thus, the numerator can be written as If we use the MAJ for both hazard functions, then there is no closed form solution. To see why this is so, note that within the exponential, the integral of a piecewise linear function is the sum of a series of quadratic functions, and the overall integral has no closed form solution. This problem motivates the piecewise mid-age joinpoint (PMAJ) model, where we use a series of piecewise constant functions to approximate the MAJ model. Figure 2 gives the PMAJ model together with the piecewise constant model used by Fay et al [1] for 70 to 90 year olds from the SEER 12, 1998–2000 rates for all invasive (first) cancer incidence rates per person-years alive. Remember, although both Figure 1 and Figure 2 plot incidence rates, we additionally need similar rate functions for mortality rates to calculate the ACPDvD. Figure 2 SEER 12 all invasive cancer incidence rates, 1998–2000, all races, both sexes: Piecewise constant and PMAJ methods. Now consider the denominator of the ACPDvD, the probability of being alive and cancer-free at age x, denoted . For reference, in Table 1 we give the notation. The only change from the notation in Fay et al [1] is that we use the subscript a to represent all causes of events instead of a blank subscript. For example, we let S*(u) = . Other notation in this paper is defined as it is introduced. Fay et al [1] assumed that the risk of death from other causes does not change if you have previously been diagnosed with cancer, then used the key result mentioned above together with some algebra and calculus to derive the denominator. Then the ACPDvD between the ages of x and y given alive and cancer-free just before age x is Table 1 Notation Random Variables and Parameters T = age at death T* = age at first cancer or death before cancer J = type of death J* = type of event  (J = d) = death from cancer  (J* = c) = first cancer  (J = o) = death from other causes  (J* = o) = death before first cancer λc(t) = rate at t for first cancer given alive = rate at t for first cancer given alive and cancer-free λo(t) = rate at t for death before cancer given alive = rate at t for death before cancer given alive and cancer-free λd(t) = rate at t for death from cancer given alive λa(t) = rate at t for death given alive = rate at t for first cancer or death before first cancer given alive and cancer-free Observations Within the age interval, [ai, ai+1), and within the calendar interval of interest we observe... ci = number of first cancer incident cases = estimate of person-years alive associated with j = c, d, o (DevCan uses the sum of mid-year populations during the calendar interval of interest) di = number of cancer deaths oi = number of other deaths The details of the MAJ and the PMAJ models are given in the next two sections. Readers only interested in the practical ramifications of the choice in models may skip to the examples and discussion section. Mid-age group joinpoint estimator In Fay et al [1], the rates were estimated by a piecewise constant model. Here we use a mid-age group joinpoint (MAJ) model, where we draw lines connecting the midpoints of the intervals except the first and last interval. The first interval is constant until the midpoint, and the last interval is constant after a nominal "midpoint". This nominal "midpoint" is half the length of the previous age interval from the beginning of the last interval, and would be the midpoint if the last age interval was the same length as the previous interval. We introduce new notation for breaking up the ages. Fay et al [1] used 0 = a0 <a1 < ··· <ak <ak+1 = ∞. Here we use a joinpoint model with joins at the midpoints (and nominal midpoint), Let (The indices start at -1 so that the index values for the rate estimators, , match up with the count notation of [1].) The MAJ estimator for the rate of event j (for j = c, d, or o) at ti (for i = 0,1,..., k) is where ji is either ci, di, or oi as defined in Table 1. (Note that , where is the piecewise constant function used in [1]). We define and . For j = a, MAJ estimator for the rate at ti is Then for t ∈ [ti, ti+1) for i = 1,..., k, we define as the point on the line defined by connecting the points (ti, ) and (ti+1, ). In other words, Where and Thus, αj,-1 = and βj,-1 = 0, and similarly by taking limits as tk+1 → ∞ then αj,k = and βj,k = 0. Now for u ∈ [ti, ti+1) is Note that (for ℓ = 0,1,..., k) so that for i = 0,1,...,k, Also notice that (when u < ∞) Therefore when u ∈ [ti, ti+1), Let (x, y) be the estimator of A(x, y) using the MAJ model. The two integrals we need to estimate for (x, y) are of the type, where in the numerator of (x, y) we need (i.e., j = c and h = a in equation 7), and in the denominator of (x, y) we need . Suppose, without loss of generality, that t ∈ [ti,ti+1), then where Rj,h(tℓ, v) (for ℓ = - 1,0,1,2,..., i and v ≤ tℓ+1) is defined implicitly (see the Appendix). Then, Piecewise mid-age group joinpoint estimator In the MAJ model we divided up the age line into k + 2 intervals. Here we define those intervals in both the ti notation and the ai notation. In the MAJ model the rates for the first and the last intervals are represented by lines with zero slope, and the rates for the ith interval (i = 1,...,k) for the jth rate type (j = a, c, d, o) is a line defined by connecting the points (ti-1, ) and (ti, ) (see equations 2 and 3 for definition of ). In the PMAJ model we divide the ith interval into mi equal sized intervals, and use a piecewise constant estimate on each of those mi intervals. One way to define mi is to chose mi so that each equal sized interval is 1/2 year long. In other words, mi = 2(ti - ti-1). This is the definition of mi that we use for the DevCan software (starting with version 5.0, see [3]), but all the following holds for arbitrary mi. In Figure 2 we show the PMAJ model with half-year intervals and the piecewise constant model for the US all invasive cancer mortality rates for ages 70 through 90 years. Here are the details. Consider the hth (for h = 1,..., mi) of the mi intervals within interval i (for i = 1,...,k) for rate type j (for j = a, c, d, o). This interval is For convenience we introduce new notation for the ends of this interval, let so that ti-1,0 = ti-1 and = ti. At the beginning of this interval the value of the rate is (see equations 4 and 6 for definitions of αj,i-1 and βj,i-1). Similarly at the end of this interval the rate is For the PMAJ model we simply assume a constant rate equal to the average of the beginning and the end values of the rate over this interval. In other words, under the PMAJ model for any t ∈ [ti-1,h-1,ti-1,h) we estimate the rate with Since the PMAJ model is a piecewise model, we can use Appendix A of [1] to express the estimator of age conditional probability of developing cancer. The only hard part is correctly defining the starting and ending of each piecewise interval. The ends of these intervals are For convenience write these interval ends with only a single index as where and m0 = 1. In other words, t-1 = τ0 and for i = 0,1,..., k, then ti= τg(i) and ti,h = τg(i)+h, where . Now we can follow very similar notation to Appendix A of [1]. We now repeat that Appendix with the modifications to notation required for the PMAJ model. Let the estimator of A(x,y) under the PMAJ model be denoted (x,y). Let τi ≤ x <τi+1 and τj <y ≤ τj+1 for x <y,i ≤ j, and j ≤ M + 2. For convenience we regroup the ages after inserting group delimiters at x and y. Let the new delimiters be 0 = b0 ≤ b1 ≤ b2 ≤ ··· ≤ bM+3 = ∞ where b0 = τ0,..., bi = τ i, bi+1 = x, bi+2 = τ i+1,..., bj+1= τ j, bj+2 = y, bj+3 = τj+1,..., bM+3 = τM+1 = ∞. We let and similarly and . In this notation, the probability of developing cancer by age y given survival until age x is A(x, y) = A(bi+1, bj+2), and under the PMAJ model we estimate it with Because or may equal zero and bℓ+1 may equal infinity, we let . These integrals are where the case λ = 0 and bℓ+1 = ∞ is one of the "impossible" hypothetical cohorts (see Section 3.1 of [1]). Thus, we obtain, Examples and discussion In this section we explore several different methods for estimating the rate functions, all using the formula of Fay et al [1] (e.g., all using equation 1). This comparison explores the differences between the piecewise constant method proposed in Fay et al [1], the PMAJ method, and the MAJ method. A different comparison emphasizing differences between versions of the DevCan software is described in the supplemental material [see Additional file 1]. For all of the examples we use data from 1998–2000 [6]. The incidence data come from the Surveillance, Epidemiology, and End Results (SEER) program of the (U.S.) National Cancer Institute, and mortality data from the (U.S.) National Center for Health Statistics. We use the SEER 12 registries which cover about 14 percent of the U.S. population. We only use the mortality data covering the same area as the SEER 12 registries cover. Because the SEER 12 registries have complete coverage only back through 1992, we only look back in the database until 1992 to delete any incident case that had previously been diagnosed with the cancer of interest. These incident cases are deleted so that they are not counted when estimating the counts of first cancer incidence (the ci values). The mid-year population estimates (the ni values) come from the sum U.S. Census estimates of mid-year populations from 1998, 1999, and 2000 for the SEER 12 catchment areas for the appropriate sex group (e.g., males for prostate cancer). In Table 2 we show the results for all invasive cancers and acute lymphocytic leukemia for both sexes, prostate cancer for males, and breast cancer for females. We see the PMAJ values approximate the MAJ values very well. In conclusion, we have described several methods for estimating rates for input into a formula to calculate ACPDvD, and we have shown that the PMAJ method provides fast and reasonable estimators for the rates. Appendix: Calculation of R function Recall that Rj,h(tℓ, v) represents an integral with 4 parameters. We can write it as To simplify notation substitute let tℓ = u and αjℓ = αj,βjℓ = bj,αhℓ = ah, and βhℓ = bh. Thus, Case 1: bj = 0 and bh = 0 For our application, whenever v → ∞ then bj = 0 and bh = 0, so this is an important special case. When bj = 0 and bh = 0 and ah = 0 and we obtain which goes to ∞ when v → ∞. When bj = 0 and bh = 0 and ah ≠ 0 and we obtain which goes to aj/ah when v → ∞. Case 2: General Case with v < ∞ To calculate the integral, R(u, v, aj, bj, ah, bh) for finite v, we can use an adaptive use of Romberg's algorithm for numeric integration (we follow closely Lange [7], pp. 210–211). Let Divide the interval [u, v] into n equal subintervals of length (v - u)/n, and let Then limn→∞ Tn = R(u, v, aj, bj, ah, bh). A more accurate approximation uses Romberg's algorithm, Let be our estimate of R. The algorithm we use to calculate is as follows: 1. Choose n. 2. Calculate Tn. 3. Calculate T2n. 4. For i = 1 to Imax do: • If then let and stop. • Otherwise calculate , and continue. For example, one could use n = 100 and δ = 10-5 and Imax = 100. Supplementary Material Additional File 1 Comparing the method of Wun, Merrill, and Feuer (1998) to the PMAJ method. We calculate lifetime risks of developing certain cancers for different race and sex combinations. For each lifetime risk we give the old method of Wun, Merrill, and Feuer [4], the PMAJ method, and the percent difference. In general, the two methods agree to within about 2 percent. Click here for file Acknowledgements I would like to thank Kathy Cronin for suggesting the PMAJ method and thank her and Ram Tiwari for reading and commenting on drafts of this article. ==== Refs Fay MP Pfeiffer R Cronin KA Le C Feuer EJ Age-conditional probabilities of developing cancer Statistics in Medicine 2003 22 1837 1848 12754719 10.1002/sim.1428 Ries LAG Eisner MP Kosary CL Hankey BF Miller BA Clegg L Mariotto A Fay MP Feuer EJ Edwards BK eds SEER Cancer Statistics Review, 1975–2000 National Cancer Institute Bethesda, MD 2003 DevCan Probability of Developing or Dying of Cancer Software, Version 5.2 Statistical Research and Applications Branch, National Cancer Institute 2004 Wum L-M Merrill RM Feuer EJ Estimating lifetime and age-conditional probabilities of developing cancer Lifetime Data Analysis 1998 4 169 186 9658774 10.1023/A:1009685507602 Merrill RM Feuer EJ Risk-adjusted cancer-incidence rates (United States) Cancer Causes and Control 1996 7 544 552 8877053 Surveillance, Epidemiology, and End Results (SEER) Program DevCan database: SEER 12 Incidence and Mortality, 1993–2000, Follow-back year = 1992 National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2003, based on the November 2002 sub mission. Underlying mortality data provided by NCHS . Lange K Numerical Analysis for Statisticians 1999 Springer:New York

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==== Front Genet Vaccines TherGenetic Vaccines and Therapy1479-0556BioMed Central London 1479-0556-2-101532445610.1186/1479-0556-2-10ResearchDurable cytotoxic immune responses against gp120 elicited by recombinant SV40 vectors encoding HIV-1 gp120 ± IL-15 McKee Hayley J [email protected]'sao Patricia Y [email protected] Maria [email protected] Puri [email protected] David S [email protected] Department of Pathology, Jefferson Medical College, Philadelphia, PA, USA2 School of Medicine. Foundation for Applied Medical Research. Division of Gene Therapy. Laboratory of Vector Development. University of Navarra. Irunlarrea 1. 31008. Pamplona. Spain2004 23 8 2004 2 10 10 24 12 2003 23 8 2004 Copyright © 2004 McKee et al; licensee BioMed Central Ltd.2004McKee et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency. Methods SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120. Results Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with ≥ 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses. Conclusions Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV. SV40HIV-1 gp120IL-15cytotoxic memory ==== Body Introduction The development of an effective vaccine against HIV has been hindered by a variety of problems. The high mutation rate of the virus itself is such that it represents a moving antigenic target during the course of an infection [1-4]. Furthermore, HLA-A and -B expression is directly downregulated by HIV (via intracellular blocking of class I MHC-export to the cell surface by HIV-1 Nef and Vpu), so that efficient antigen presentation is compromised [1,5]. Compared to administration of protein antigen or naked DNA, an infectious vector could be more effective at enhancing antibody and cytotoxic responses against a transgene product. Application of such a strategy, however, has been often complicated by the development of neutralizing immune responses, principally antibodies, against vector coat antigens [6-10]. These neutralizing antibodies arise because the viral vectors enter cells largely through endocytic pathways. Their capsids, like most other particulate antigens, are processed at the time of infection and presented to the immune system. Resulting immune responses neutralize subsequent injections of the vector, and so limit the ability of that vector to be used repeatedly to boost immune responses. This limitation can be circumvented by repeatedly changing the serotype of the antigen-carrying vector, or by using recombinant Tag-deleted SV40-derived gene delivery vectors (rSV40s) for immunization. Several studies have shown, both directly and indirectly, that rSV40 vectors do not elicit detectable neutralizing antibodies [11-13]. Even repeated administration of single [11,12] or different [13] rSV40 vectors in normal, immunocompetent hosts does not generate antibodies against the vector capsid proteins sufficiently to impair the ability of these vectors to deliver their genes efficiently in vivo. The explanation for this unusual state of affairs may lie in the fact that SV40 enters cells via caveolae and thence travels directly to the nucleus, bypassing cellular antigen processing [14-16]. Thus, only proteins expressed by virus can elicit immune responses. Since, for Tag-deleted rSV40 vectors (unlike wild type SV40), capsid proteins are not expressed, immune responses can only be generated by transgene products [11,12]. Whether for this or for other reasons, rSV40 vectors can be used multiple times to prime and/or boost immune responses against antigens encoded by the transgenes they carry [13,14]. We have previously shown that powerful transgene-specific cytolytic and serum antibody responses can be detected in mice inoculated with rSV40 carrying the cDNA for SIVmac239 envelope glycoprotein gp130 [12]. Four to five monthly immunizations were adequate to produce >50% specific lysis of envelope-expressing target cells, even with effector:target ratios of 10:1 [12]. Other investigators have reported that co-administration of vectors carrying immunostimulatory cytokines was useful in augmenting anti-lentiviral immune responses [17-19]. IL-15 has various immunostimulatory and immunomodulatory effects, among which is the ability to upregulate activated T cell proliferation and induce cytotoxic T cell activity [20]. It also promotes cytotoxic T cell memory [21,22]. Both antibody and cell-mediated immune responses may be useful to protect from HIV infection and progression to AIDS [23-26]. However, there is a particularly good correlation between long-term non-progression to AIDS and strong CTL responses in HIV-positive individuals [22,27-31]. Weak CTL responses are generally seen in those who progress rapidly to disease, and in children. Because of the importance of a virus-specific cytotoxic T cell (CTL) response, one of the major aims of any vaccine should be to elicit strong HIV-specific CTL responses [32,33]. We used rSV40s to study the generation and longevity of both humoral and cell-mediated responses in an effort to generate immune responses against the HIV-1 envelope glycoprotein, gp120. We also tested whether co-immunization regimens involving rSV40 delivery of both IL-15 and gp120 augmented and/or accelerated SV40-mediated immune responses further. Methods Cell Lines The murine mastocytoma cell line P815 (ATCC, Bethesda, MD, USA) was used, and maintained in culture with Dulbecco's Modified Eagle's medium (DMEM), supplemented with 10% newborn calf serum (NCS) (Gibco BRL/Life Technologies, Grand Island, NY, USA). COS-7 cells (ATCC, Bethesda, MD, USA), were used to expand stocks of recombinant SV(gp120) and SV(mIL-15) viruses. Cytotoxic lymphocytes were obtained from spleens of immunized mice, and cultured in RPMI-1640 (Gibco BRL/Life Technologies, Grand Island, NY, USA) supplemented with 10% NCS (RPMI-10). Rabbit kidney fibroblasts (RK13 cells) and CV-1 cells (African green monkey kidney cells) were obtained from ATCC (Bethesda, MD, USA). RK13 cells were used to propagate stocks of VCB41, a vaccinia virus vector carrying HIV-1NL4-3 envelope gp120 sequence. Mice BALB/cJ mice aged 6–8 weeks were purchased from Jackson Laboratories, Bar Harbor, ME, USA. They were fed and housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. Use of mice in the laboratory protocols described was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. Generation of SV(gp120) A 1.6 kb DNA fragment encoding gp120 from HIV-1NL4-3 was made by PCR using primers with engineered restriction sites. This PCR product was cloned into pT7A5 (a plasmid containing an SV40 genome, in which large T antigen gene was replaced by cytomegalovirus (CMV) immediate early promoter and downstream polylinker), giving pT7A5-gp120. To make SV(gp120), the SV(gp120) genome was released from the carrier plasmid by restriction digestion, and used to make recombinant virus in COS-7 cells as described previously [34]. Virus stocks were purified and titered, as described elsewhere [34]. SV(HBS), a control virus for these studies, carries hepatitis B surface antigen (HBsAg), and has been reported previously [11]. Generation of SV(mIL-15) To generate a recombinant SV40 virus with the murine IL-15 transgene (SV(mIL-15)), mIL-15 cDNA was cloned into pSL-4p, which contains a Tag-deleted SV40 genome [[35], Vera, et al., in preparation], to yield prSVmIL-15. Virus was made from this plasmid in COS-7 cells as previously reported [32]. Immunization of mice Mice were given monthly 1 × 109 infectious units (IU) of SV(gp120) ± SV(mIL-15) intraperitoneally (IP). In some studies, final administrations included both IP and subcutaneous (SQ) inoculations. SV(HBS) was used as a control antigen-carrying vector. Specific immunization schedules are described in the Results section, below. Stably-transfected P815 target cells for cytotoxicity assays Production of HIV Env-expressing stably transfected targets is similar to the procedure used for generating SIV Env-expressing targets [12]. Briefly, gp120 cDNA was cloned into pCDNA3. The resulting plasmid, pcgp120, was co-transfected into P815 cells together with the neomycin resistance-carrying plasmid, pSV2Neo. Transfected cells were selected in G418-supplemented DMEM-10, then cloned by limiting-dilution. Viable clones were expanded, assayed for gp120 expression by flow cytometry, and maintained thereafter in G418-supplemented medium. Flow cytometric detection of cell surface gp120 expression Flow cytometry was used to verify gp120 expression on the surface of P815 cells. A recombinant vaccinia virus carrying HIV-1NL4-3 gp120 (VCB41, NIH AIDS Reference Reagent Repository Program (NIH-ARRRP)) was used both as a positive control for gp120 expression and also to generate gp120-expressing target cells in some experiments. Cells that had been stably-transfected with plasmid gp120, or infected with VCB41 both expressed gp120, as assayed by flow cytometry (Coulter-Epic, Kimmel Cancer Center, TJU) (data not shown). The gp120-expressing P815 population was then cloned by limiting dilution. Clonal outgrowths were then reanalyzed by cytofluorimetry (FACS, data not shown) and the single clone expressing the highest levels of gp120, clone 24, was used in subsequent studies as a target for cytotoxicity assays. Anti-gp120 binding-antibody detection using a CELISA An ELISA method was used to assay the activity of anti-gp120 antibodies elicited by immunization of the mice with SV(gp120) ± SV(mIL-15). The strategy for our appraoch to testing for antibodies vs. HIV Env is similar to one we have used to measure binding activity vs. SIV Env [12]. Briefly, a cell-based assay was developed using VCB41-infected P815 cells (cells were infected with virus for 48 hours prior to being used in assay) as control targets. Sera were taken from mice at 2- and 4-week intervals after immunization(s). Antibody reactivity vs. cell membrane-expressed gp120 was tested by measuring A405nm of test sera vs. VCB41-infected P815 cells, subtracting A405nm due to binding to wild type (wt) VV-infected P815 cells, and also subtracting A405nm of control sera from mice immunized in parallel with a control rSV40, SV(HBS) Measurement of cytotoxic lymphocyte activity by specific lysis of 51Cr-labeled target cells Wild type (wt) P815 cells, or clone 24 P815 cells expressing gp120 were the target cells for unselected lymphocytes from spleens of mice immunized with SV(gp120) (± SV(mIL-15)), or SV(HBS) as control. Where SV(gp120) was used alone, P815 cells infected with wt vaccinia virus or VCB41 were target cells for spleen cells of immunized mice. Mice were boosted simultaneously with 1 × 109 IU intraperitoneally (IP) and 1 × 108 IU subcutaneously (hind footpads) usually 4 d before assay, but up to 1 month prior to assay, to test cytolytic lymphocyte memory. Spleen cell concentrations adjusted to 2 × 106 / ml with RPMI-10. In some assays of cytotoxic lymphocyte memory, effector cells were harvested 1 month after the final injection. Effector cells from immunized mice were prepared as described, but in addition, were incubated with 5 μg/ml Concanavalin A (Con A, Sigma Chemical Co., St. Louis, MO) overnight, prior to assay. Con A-stimulated cells were then harvested, washed once in RPMI-10, and then used with target cells in the assay. P815 target cells were washed, then labeled with 51Cr (ICN Biomedicals, Inc., Irvine, CA, USA) (100 μCi per 1 × 106 cells) at 37°C, 5% CO2 for 4 h as described previously [12]. Afterwards, target cells were washed, then plated in triplicate with effector cells (splenocytes) at effector:target (E:T) ratios of 20:1 and 10:1, and incubated at 37°C, 5% CO2 for 4 hours. Supernatant 51Cr was counted (1282 Compugamma CS, LKB) [12]. Mean specific lysis was calculated as: Mean c.p.m. for gp120-immunized effector cells mixed with gp120-expressing targets, minus the mean c.p.m. control (SV(HBS))-immunized effector cells vs gp120-expressing targets, and expressed as a percentage of the maximal target cell lysis (target cells incubated with 1% Triton-X). Background release of 51Cr from wild type target cells was subtracted. Thus: % specific 51Cr release = {[c.p.m. 51Cr released by SV(gp120)-immune populations from gp120-expressing P815 cells] minus [c.p.m. 51Cr released by SV(HBS)-immune lymphocytes from gp120-expressing P815 cells]}, divided by [c.p.m. 51Cr released by Triton X-100 from gp120-expressing P815 cells]. The same calculations were done for lysis of wild type P815 cells by gp120-immune and control-immune effector populations. These numbers were then subtracted from the calculated 51Cr release above to determine the gp120-specific lysis of target cells by SV(gp120)-immunized effector cells. Western analysis of IL-15 expression in SV(mIL-15)-transduced P815 cells P815 cells were transduced with SV(mIL-15) ×1 at m.o.i. = 100. Culture supernatants were harvested at several times post-transduction, and stored at -80°C. At day 6 post-transduction, a well of cells was harvested and lysed (2% NP40, 50 mM Tris pH7.4,150 mM NaCl, 1 mM EDTA, 10% Glycerol + protease inhibitor cocktail (25× stock Complete™ EDTA-free protease inhibitor cocktail, Roche Diagnostics GmbH, Mannhein, Germany)). Remaining wells were activated non-specifically with 5 mg/ml Con A, and supernatants harvested at various times thereafter. 3 days after con A stimulation, cells were lysed as described above. 50 μg of each culture supernatant or lysate were loaded on a 4–20% Tris-HCl gradient gels (Ready Gel, Bio-Rad, Hercules, CA, USA). 50 ng recombinant human IL-15 was used as a positive control. Samples were electrophoresed, and blotted to PVDF membranes (Immobilon™-P, Millipore Corporation, Bedford, MA). Blots were blocked overnight at 4°C with 5% milk in PBS + Tween-20 (0.05%). Rabbit anti-mouse IL-15 (Abcam, Cambridge, UK) was used as primary antibody, (diluted 1:500 with PBS-Tween), for 2 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was used at 1:10,000 dilution in PBS-Tween, for 1 h at room temperature. Signal was detected with chemiluminescence reagent (ECL Plus, Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK,) Assaying for IFNγ production stimulated by IL-15 COS-1 cells were infected with SV(mIL15) or SVLUC (carrying luciferase) as a negative control. 24 h later, the media were changed and cells incubated 48 h in 500 μl of RPMI 10% serum/well. Fresh mouse spleen cells (5000/well) then cultured 48 h with 100 μl of cell supernatant + 100 μl of RPMI 10% serum. IFNγ ELISA (Pharmingen) was performed on the supernatant from these cultures. Results Stably transfected HIV-1 gp120-expressing P815 cells P815 cells stably transfected to express HIVNL4-3 gp120 were selected and cloned by limiting dilution (see Methods). We used flow cytometry to identify the clone most strongly positive for cell membrane gp120. Compared to other stably-transfected clones, "clone 24" expressed gp120 at the cell membrane best (data not shown). VCB41-infected and SV(gp120)-transduced P815 cells also expressed substantial cell membrane gp120. Control wtP815 cells, or P815 cells infected with wt VV did not (data not shown). Therefore, clone 24 cells were used to assay gp120-specific immune responses. In both antibody and cytotoxicity assays, two different types of background were subtracted from the responses of gp120-immunized mice: serum binding or cellular reactivity from gp120-immunized animals vs. wt P815 cells and reactivity from control (i.e., SV(HBS))-immunized rSV40-immunized mice vs. clone 24 cells. Thus, data presented below reflect gp120-specific responses against clone 24. Immunization with SV(gp120) Normal BALB/c mice were inoculated with SV(gp120), and their sera were assayed for reactivity vs. gp120 by CELISA. The details of this cell-based ELISA, or CELISA, as described in Methods. Specific binding antibody activity was first statistically significant, compared to prebleed sera 2 weeks after the second inoculation of SV(gp120) (P = 0.000332, using two-tailed Student's t-test) and reached a plateau after the third inoculation (P = 0.000000316 by the same analysis) (Figure 1). Additional immunizations beyond the third did not further increase detectable antibody levels (data not shown). Figure 1 Serum antibody against HIV-1NL4-3 envelope glycoprotein gp120 in mice receiving multiple inoculations of SV(gp120) BALB/cJ mice were immunized at monthly intervals with 1 × 109 infectious units (IU) SV(gp120), IP. They were bled biweekly. Gp120-specific antibody reactivity was assayed by CELISA, as described in Methods and in reference #12. Specific binding of HIV-1 Env is shown here as specific A405nm, ± S.E.M. Cytolytic responses against gp120: testing for cytotoxic lymphocyte memory An effective anti-lentiviral immunization regimen should generate cytotoxic memory cells. To see if SV(gp120) treatment could do this, mice were immunized once with SV(gp120) IP, then sacrificed 1 month later, without further treatment. In order to lyse target cells, committed cytotoxic cells require activation. However, to avoid antigen-specific selection and specific stimulation only of gp120-reactive cytotoxic cells, splenic lymphocytes were non-specifically stimulated by overnight incubation with Con A. A single immunization with SV(gp120) alone elicited only weak memory lytic responses (≤ 10% specific lysis) against gp120-expressing target cells (Figure 2). Figure 2 Specific cytolytic activity against HIV-1NL4-3 gp120 in mice immunized with SV(gp120) and assayed one month after final injection. BALB/cJ mice were immunized twice with SV(gp120) IP at monthly intervals. Splenocytes were harvested one month after final inoculation. Unselected effector cells were added to 51Cr-labelled target cells and specific lysis of gp120-expressing cells was calculated as described in Methods, ± S.E.M. Results shown here represent ≥ 3 independent determinations per data set. A second group of animals received a second inoculation with SV(gp120) one month after the first, then were assayed the same way one month later for anti-gp120 cytolytic activity. These mice made stronger specific memory responses (15–20% specific lysis) than did animals given only a single inoculation (P ≤ 0.04, by Student's t-test, comparing 2 injections with just one) (Figure 2). To test whether SV(gp120) could elicit very long term cytotoxic lymphocyte memory, mice were immunized monthly ×8 with SV(gp120) IP. A final IP inoculation with SV(gp120) was given 1 year after their eighth immunization. They were sacrificed 4 days later, and direct gp120-specific splenocyte cytotoxicity was measured. Unselected spleen cells from all animals made very strong (≥ 50% specific lysis) gp120-specific cytolytic responses (mean specific lysis of 61% ± 4.2). IL-15 expression and secretion in SV(mIL-15)-transduced P815 cells Because higher levels of durable memory cytotoxic responses could be achieved with repeated injections of SV(gp120), and lower levels were seen with 2 injections, we tried to accelerate development of such responses using IL-15, delivered by transduction. To determine if IL-15 could be expressed by transduction, P815 cells were transduced with SV(mIL-15) at m.o.i. = 100. Culture supernatants were harvested 36, 72 and 144 hours later, at which point the cultured cells were activated with Con A. Culture supernatants were collected at 24 and 72 hours post-activation. Supernatants were assayed for IL-15 secretion by Western analysis as described in Methods, using rabbit antibody vs. murine IL-15 (Figure 3). The positive control, recombinant human IL-15, has approximately 60% sequence homology with murine IL-15). IL-15 secretion was detectable, but just barely so, in unstimulated culture supernatants. It was abundant by 72 hrs post-stimulation. These data were used in planning co-transduction experiments. Figure 3 Western analysis of IL-15 expression in culture supernatants from SV(mIL-15)-transduced P815 cells P815 cells were transduced with SV(mIL-15) at a m.o.i. = 100. 1.5, 3 and 6 days post-transduction, culture supernatants were collected. Cultures were then stimulated with Con A and additional supernatants harvested 24 and 72 hours later. IL-15 secretion into supernatants was visualized by Western analysis. Recombinant human IL-15 (rhIL-15) was the positive control (non-adjacent lane). Supernatants from unstimulated cultures were tested in parallel (non-adjacent lane). The functionality of the IL-15 produced in this fashion was tested by exploiting the ability of IL-15 to elicit production of IFN-γ by lymphocytes. Thus, CV-1 and COS-7 cells (African green monkey kidney cells) were transduced by SV(mIL-15), then cultured for 72 hrs. Control cultures of the same cells were transduced with SVLUC (carrying luciferase as a transgene). Normal mouse spleen cells were cultured for 42 hrs in 200 μl of the resulting culture supernatants. Production of IFN-γ by the spleen cells was measured by ELISA. Supernatants from COS-7 and CV-1 cells elicited respectively 2056 ± 363 pg/ml and 880 ± 196 pg/ml IFN-γ. Supernatants from SVLUC-transduced cells did not elicit detectable interferon secretion by spleen cells (<20 pg/ml). Effects of IL-15 in cytotoxic lymphocyte responses against gp120 To determine whether coordinate administration of SV(mIL-15) plus SV(gp120) improved cytolytic responses against gp120, mice were given two sets of injections IP, one month apart. Normal BALB/c mice received IP with 109 IU of rSV40: one group was given SV(mIL-15) alone, and one group SV(gp120) alone. Three other groups received both SV(mIL-15) and SV(gp120): either SV(mIL-15) followed 3 days later by SV(gp120), or SV(gp120) first, followed by SV(mIL-15). The final group was given both SV(mIL-15) and SV(gp120) simultaneously. The 3 day separation between the two vectors was used because of the strength of the signal for secreted IL-15 by Western blotting at 72 hours post-stimulation (see above). One month later, unselected spleen cells were assayed as described above for cytolytic activity against gp120-expressing clone 24 cells. Adding IL-15 to the immunization regimen greatly increased gp120-specific cytolytic responses (Figure 4). Also, the timing of cytokine administration relative to SV(gp120) inoculation significantly affected the responses seen. Mice given SV(mIL-15) 3 d after SV(gp120) did not make detectable gp120-specific cytotoxic responses. Simultaneous inoculation with SV(mIL-15) and SV(gp120), however, increased specific cytolysis to ≥ 20%, which was significant at E:T = 20:1 (P ≤ 0.05, using Student's t-test) compared to SV(gp120) alone. The most dramatic results were observed when SV(mIL-15) was administered 3 d before SV(gp120). Those mice demonstrated highly significant augmentation by SV(IL-15) of gp120-specific lysis, which was ≥ 60% at both 20:1 and 10:1 E:T ratios (P ≤ 0.02 using Students' t-test, compared with immunization of SV(gp120) alone). Mice injected ×2 with SV(mIL-15) alone made no significant gp120-specific cytolytic responses at either 20:1 or 10:1 effector:target ratios. Mice given only SV(gp120) demonstrated ≈ 10% specific lysis at E:T = 20:1. Figure 4 Specific cell-mediated responses against gp120-expressing target cells by splenic effectors from co-immunized mice Mice were given two monthly injections with either SV(mIL-15), SV(gp120) or both cytokine and antigen sequentially or simultaneously IP. One month after the final inoculation(s), unselected spleen cells were assayed for specific cytolytic activity against gp120-expressing clone 24 cells labeled with 51Cr, as described in Methods. Results shown here represent ≥ 3 independent determinations per data set. Effect of IL-15 co-administration on anti-gp120 antibody responses Mice receiving SV(mIL-15) and/or SV(gp120) (or the control vector, SV(HBS)) according to the schedules outlined above were tested to determine the effect, if any, of such co-administration in anti-gp120 serum antibody responses. CELISA and calculation of gp120-specific antibody binding were performed as described in Methods. Slight binding antibody activity was detected 2 weeks after the first inoculation(s), in all SV(gp120) recipient groups. Levels of SV(gp120)-induced groups made detectable antibody responses were not appreciably affected by coadministration of SV(mIL-15) (data not shown). Discussion In this study, we used rSV40 vectors to elicit HIV-1NL4-3 gp120-specific cytotoxic lymphocyte and antibody responses. We have observed that these vectors may be administered repeatedly to boost those responses. Further studies also suggested that such responses are durable in vivo. Our results here demonstrate several important strengths of using rSV40 vectors to immunize against lentiviral antigens: Among these are the ability of the vector to be administered multiple times without eliciting neutralizing responses [11-13], and the magnitude of the cytotoxic responses to the vector-encoded lentiviral target antigen. When SV(gp120) was given alone, i.e. without added SV(mIL-15), levels of specific cytotoxicity increased with additional SV(gp120) injections: After 2 injections, ≈ 20% specific lysis was seen, which increased to >70% specific lysis after 7 injections. The potency of rSV40 immunization to elicit cytotoxic immune responses is underscored by the fact that these responses were measured in direct 51Cr-release assays: unselected lymphoid organ populations were added directly to labeled target cells at low E:T ratios, and specific 51Cr release was measured. Analysis to confirm CD8 expression, or expression of other CTL markers was not performed on the effector cells. However, it is unlikely that these data reflect the cytotoxic activity of NK cells. NK cytolytic activity is non-specific and does not increase with repeated immunization. The patterns of 51Cr release observed in the current studies were extensively controlled to ascertain the antigen-specificity of the cytolysis observed: background lysis of wild type P815 cells was subtracted, as was lysis by lymphocytes from mice immunized with an irrelevant rSV40 vector. We also found that cytolysis increased with increasing numbers of immunizations, which is not a characteristic of NK cell-mediated lysis. Since a key goal for a vaccine against HIV is to generate immune responses that are durable in vivo, we tested whether cytotoxic lymphocyte activity elicited by SV(gp120) immunization, was detectable one month after inoculation. Thus, cytolytic responses, assayed one month after a second injection, were ≈ 20%, which is comparable to those of splenic cytotoxic cells assayed four days following a third inoculation (data not shown). Further, mice given multiple injections of SV(gp120), then rested for one year, gave ≈ 70% specific lysis when challenged with SV(gp120). Therefore, SV(gp120) administration may thus favor development of cytotoxic lymphocyte memory. In an attempt to accelerate and to improve upon these specific cytotoxic and particularly cytotoxic memory responses, we co-immunized mice with SV(gp120) and a rSV40 carrying mouse IL-15. IL-15 promotes cytotoxic lymphocyte responses, and in particular, cytotoxic memory responses [23,24]. The biological effects of IL-15 are less well understood than are those of some of the other immunostimulatory cytokines that have been applied to these types of immunization protocols, such as IL-2, IL-12 and IFN-γ. IL-15 is not a T cell-derived product, but rather appears to be produced by a variety of cells, such as epithelial cells, stromal cells and muscle. It acts on activated T cells, sometimes similarly to IL-2, but it has activities distinct from those of IL-2. IL-15 may play a role in T cell activation in the CNS. It also promotes cytotoxic responses, cytotoxic T cell memory, and natural killer (NK) cell maturation [33,34]. Accordingly, our analysis of the contribution by IL-15 to cytotoxic responses, focused mainly on the ability of SV(mIL-15) to augment specific cytotoxic responses of spleen cells from animals rested 1 month following immunization. Because quiescent cytotoxic T cells are not strong effectors, we non-specifically activated the splenocytes prior to assay with Con A. Non-specific activation was used to avoid specifically enriching effector cell population for gp120-specific cells in vitro. Furthermore, low effector:target ratios (20:1 and 10:1) were used in these assays. Our immunization protocols tested both simultaneous and staggered administration of rSV40s carrying HIV-1NL4-3 gp120 and murine IL-15. IL-15 co-immunization dramatically accelerated cytotoxic responses, depending on the immunization regimen used: Animals given SV(mIL-15) alone made no gp120-specific cytolytic responses. Mice receiving 2 treatments with a mixture of SV(gp120) and SV(mIL-15) gave much higher specific lysis, depending on the coadministration regimen, as compared to those receiving SV(gp120) alone (≈ 10% specific lysis). Thus, among mice given staggered injections of SV(mIL-15) and SV(gp120), the order of cytokine administration greatly affected the response: if SV(gp120)was given first, no detectable gp120-specific cytolysis was observed. However, if the cytokine was given first, followed 3 days later by SV(gp120), ≥ 60% specific lysis was seen at both 20:1 and 10:1 effector:target ratios. Why the order of cytokine administration should affect antigen-specific responses so dramatically is not yet clear. Cytokine given after, or together with antigen, may have insufficient time to augment cytotoxic responses. In addition, Western analysis of IL-15 production showed that IL-15 secretion was not detectable in supernatants beyond 36 hours, but could be stimulated subsequently. Thus, a specific, possibly brief, window for IL-15 expression and secretion may need to be attained, in order for its effects on gp120-specific responses to be detectable. We observed very high levels of specific lysis by these unselected effector populations following just two tandem injections of SV(mIL-15) followed by SV(gp120). The strong anti-lentiviral cytolytic responses we report were observed in a strain of mouse, BALB/cJ, that generally mounts relatively weak type 1 T cell responses. The finding of >60% cytolysis with two administrations of SV(gp120) + SV(mIL-15), suggests that a strategy similar to that described herein may be helpful in individuals who would generate relatively low cytolytic responses. Serum antibody levels assayed by CELISA where SV(gp120) was administered alone, multiple times, were detectable after two immunizations, and continued to increase up to week 4 following the third immunization. These responses were not further enhanced by subsequent boosting immunizations. While specific antibody responses against gp120 were detected in all experimental groups following SV(gp120) and SV(mIL-15) co-immunization, IL-15 co-administration did not augment anti-gp120 antibody levels, compared to gp120 alone. This was to be expected, since IL-15 reportedly acts primarily on T cell and NK cell functions, rather than on humoral immune responses. Our data argue in favor of using IL-15 as an adjuvant for antigen-specific immune responses, particularly cytotoxic lymphocyte responses. We also demonstrate that a single transgene, administered multiple times (>3), may be very effective at eliciting both humoral and cell-mediated responses. These results thus both corroborate and extend our previous observations [13,14,32], and suggest that combining rSV40s encoding antigens and immunostimulatory cytokines sequentially in multi-administration regimens may provide high levels of long-lasting immunity against the target antigen. Conclusions Recombinant SV40-derived gene-delivery vectors, being transparent to the immune system, can be given multiple times to prime and boost immune responses against the delivered antigens. Anti-vector immunity does not overwhelm responses against the target antigens. As well, these vectors elicit very high levels of antibody, and especially cell-meditated immunity. Finally, combining the delivery of rSV40s bearing antigens with those bearing cytokines such as IL-15 can enhance levels of immunity, particularly long-term immunity. Clearly, much work remains. However, this approach offers promise as a strategy to immunize against pathogens for which classical approaches have not been adequately effective. List of Non-Standard Abbreviations Used Table 1 Abbreviation Meaning CELISA cell-based ELISA E:T effector cell:target cell ratio gp120 major HIV envelope glycoprotein, 120 kDa HBS hepatitis B surface antigen IFNγ interferon-gamma mIL-15 mouse interleukin-15 NIH-ARRRP National Institutes of Health, AIDS Research Reference Reagent Program pNPP p-nitrophenyl phosphate VCB41 strain of vaccinia virus carrying gp120 coding sequences wt wild type Competing Interests None declared. Authors' Contributions HJM devised all the assay systems for cell- and antibody-mediated immunity against lentiviral antigens, performed all the immunization studies and assays. HM also wrote this manuscript. PYT generated the SV(gp120) construct. MV and PF generated the SV(mIL-15) and SVLUC constructs described here and performed the ELISA for IFNγ stimulated by SV(mIL-15). DSS is the Principal Investigator for this work, oversaw and planned the experimental strategies, worked with HJM in interpreting the experimental data and writing the manuscript. Acknowledgements The authors would like to thank Drs. Jean Boyer, Scott Cairns, Judy Lieberman, David Weiner and John Zaia, and the late Nava Sarver for their advice. This work was supported by grants AI46253 and AI48244. ==== Refs Swann SA Williams M Story CM Bobbitt KR Fleis R Collins KL HIV-1 Nef blocks transport of MHC class I molecules to the cell surface via a PI 3-kinase-dependent pathway Virology 2001 282 267 277 11289809 10.1006/viro.2000.0816 Graziosi C Soudeyns H Rizzardi GP Bart P-A Chapuis A Pantaleo G Immunopathogenesis of HIV infection AIDS Res Hum Retrovir 1998 14 S135 S142 9672230 McMichael A T cell responses and viral escape Cell 1998 93 673 676 9630211 10.1016/S0092-8674(00)81428-2 Haynes BF HIV vaccines: where we are and where we are going Lancet 1996 348 933 937 8843815 10.1016/S0140-6736(96)09339-7 Cohen GB Gandhi RT Davis DM Mandelboim O Chen BK Strominger JL Baltimore D The selective downregulation of class I major histocompatability complex proteins by HIV-1 protects HIV-infected cells from NK cells Immunity 1999 10 661 671 10403641 10.1016/S1074-7613(00)80065-5 Belshe RB Stevens C Gorse GJ Buchbinder S Weinhold K Sheppard H Stablein D Self S McNamara J Frey S Flores J Excler JL Klein M Habib RE Duliege AM Harro C Corey L Keefer M Mulligan M Wright P Celum C Judson F Mayer K McKirnan D Marmor M Woody G and the HIVNET Safety and immunogenicity of a canarypox-vectored human immunodeficiency virus Type 1 vaccine with or without gp120: a phase 2 study in higher- and lower-risk volunteers J Infect Dis 2001 183 1343 1352 11294665 10.1086/319863 Ramirez J-C Gherardi MM Rodriguez D Esteban M Attenuated modified vaccinia virus Ankara can be used as an immunizing agent under conditions of preexisting immunity to the vector J Virol 2000 74 7651 7655 10906221 10.1128/JVI.74.16.7651-7655.2000 Molinier-Frenkel V Gahery-Segard H Mehtali M Le Boulaire C Ribault S Boulanger P Tursz T Guillet JG Farace F Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes J Virol 2000 74 7678 7682 10906225 10.1128/JVI.74.16.7678-7682.2000 Allen TM Vogel TU Fuller DH Mothe BR Steffen S Boyson JE Shipley T Fuller J Hanke T Sette A Altman JD Moss B McMichael AJ Watkins DI Induction of AIDS virus-specific CTL activity in fresh unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen J Immunol 2000 164 4968 4978 10779808 Salmon-Ceron D Excler JL Finkielsztejn L Autran B Gluckman JC Sicard D Matthews TJ Meignier B Valentin C El Habib R Blondeau C Raux M Moog C Tartaglia J Chong P Klein M Milcamps B Heshmati F Plotkin S Safety and immunogenicity of a live recombinant canarypox virus expressing HIV type 1 gp120 MN MN tm/gag/protease LAI (ALVAC-HIV, vCP205) followed by a p24E-V3 MN synthetic peptide (CLTB-36) administered in healthy volunteers at low risk for HIV infection AIDS Res Hum Retrovir 1999 15 633 645 10331442 10.1089/088922299310935 Kondo R Feitelson MA Strayer DS Use of SV40 to immunize against hepatitis B surface antigen: Implications for the use of SV40 for gene transduction and its use as an immunizing agent Gene Ther 1998 5 575 582 9797861 10.1038/sj.gt.3300623 McKee HJ Strayer DS Immune responses against SIV envelope glycoprotein using recombinant SV40 as a vaccine delivery vehicle Vaccine 2002 20 3613 3625 12297408 10.1016/S0264-410X(02)00243-8 Sauter BV Parashar B Chowdhury NR Kadakol A Ilan Y Singh H Milano J Strayer DS Chowdhury JR A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats Gastroenterology 2000 119 1348 1357 11054394 Norkin LC Simian virus 40 infection via class I molecules and caveolae Immunol Rev 1999 168 13 22 10399061 Pelkmans L Kartenbeck J Helenius A Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nature Cell Biol 2001 3 473 483 11331875 10.1038/35074539 Pelkmans L Puntener D Helenius A Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae Science 2002 296 535 539 11964480 10.1126/science.1069784 Moore AC Kong WP Chakrabarti BK Nabel GJ Effects of antigen and genetic adjuvants on immune responses to human immunodeficiency virus DNA vaccines in mice J Virol 2002 76 243 250 11739689 10.1128/JVI.76.1.243-250.2002 Barouch DH Letvin NL Cytokine-induced augmentation of DNA vaccine-elicited SIV-specific immunity in rhesus monkeys Dev Biol (Basel) 2000 104 85 92 11713828 Bradney CP Sempowski GD Liao H-X Haynes BF Staats HF Cytokines as adjuvants for the induction of anti-human immunodeficiency virus peptide immunoglobulin G (IgG) and IgA antibodies in serum and mucosal secretions after nasal immunization J Virol 2002 76 517 524 11752142 10.1128/JVI.76.2.517-524.2002 Geginat J Sallusto F Lanzavecchia A Cytokine-driven proliferation and differentiation of human naïve, central memory and effector memory CD4+ T cells J Exp Med 2001 194 1711 1719 11748273 10.1084/jem.194.12.1711 Sharif-Askari E Fawaz LM Tran P Ahmad A Menezes J Interleukin 15-mediated induction of cytokine effector cells capable of eliminating Epstein-Barr virus-transformed/immortalized lymphocytes in culture J Natl Cancer Inst 2001 93 1724 1732 11717333 10.1093/jnci/93.22.1724 Yajima T Nishimura H Ishimitsu R Watase T Busch DH Pamer EG Kuwano H Yoshikai Y Overexpression of IL-15 in vivo increases antigen-driven memory CD8+ T cells following a microbe exposure J Immunol 2002 168 1198 1203 11801655 McMichael AJ HIV. The immune response Curr Opin Immunol 1996 8 537 539 8794022 10.1016/S0952-7915(96)80043-4 Burton DR A vaccine for HIV type 1: The antibody perspective Proc Natl Acad Sci USA 1997 94 10018 10023 9294155 10.1073/pnas.94.19.10018 Johnson RP Siliciano RF McElrath MJ Cellular immune responses to HIV-1 AIDS 1998 12 S113 S120 9632992 Pitcher CJ Quittner C Peterson DM Connors M Koup RA Maino VC Picker LJ HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression Nature Med 1999 5 518 525 10229228 10.1038/8400 Autran B Hadida F Haas G Evolution and plasticity of CTL responses against HIV Curr Opin Immunol 1996 8 546 553 8794018 10.1016/S0952-7915(96)80045-8 Schmitz JE Kuroda MJ Santra S Sasseviolle VG Simon MA Lifton MA Racz P Tenner-Racz K Dalesandro M Scallon BJ Ghrayeb J Forman MA Montefiori DC Rieber EP Letvin NL Reimann KA Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes Science 1999 283 857 860 9933172 10.1126/science.283.5403.857 Borrow P Lewicki H Hahn BH Shaw GM Oldstone MBA Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection J Virol 1994 68 6103 6110 8057491 Rinaldo C Huang XL Fan ZF Ding M Beltz L Logar A Panicali D Mazzara G Liebmann J Cottrill M High levels of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte activity and low viral load are associated with lack of disease in HIV-1-infected long-term nonprogressors J Virol 1995 69 5838 5842 7637030 Addo MM Altfeld M Rosenberg ES Eldridge RL Philips MN Habeeb K Khatri A Brander C Robbins GK Mazzara GP Goulder PJR Walker BD and the HIV Controller Study Collaboration The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals Proc Natl Acad Sci USA 2001 98 1781 1786 11172028 10.1073/pnas.98.4.1781 Addo MM Yu XG Rathod A Cohen D Eldridge RL Strick D Johnston MN Corcoran C Wurcel AG Fitzpatrick CA Feeney ME Rodriguez WR Basgog N Draenert R Stone DR Brander C Goulder PJ Rosenberg ES Altfeld M Walker B Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load J Virol 2003 77 2081 2092 12525643 10.1128/JVI.77.3.2081-2092.2003 Newman MJ Heterologous prime-boost vaccination strategies for HIV-1: augmenting cellular immune responses Curr Opin Investig Drugs 2002 3 374 378 12054082 Strayer DS Kmiec EB Effective gene transfer using viral vectors based on SV40 in Methods in Molecular Biology, Gene Targeting Vector Protocols 1999 133 Humana Press, New York 61 74 10.1385/1-59259-215-5:61 de la Luna S Martin J Portela A Ortin J Influenza virus naked RNA can be expressed upon transfection into cells co-expressing the three subunits of the polymerase and the nucleoprotein from simian virus 40 recombinant viruses J Gen Virol 1993 74 535 539 8383187 Seder RA Grabstein KH Berzofsky JA McDyer JF Cytokine interactions in human immunodeficiency virus infected individuals: roles of interleukin (IL)-2, IL-12 and IL-15 J Exp Med 1995 182 1067 1077 7561680 10.1084/jem.182.4.1067 Giri JG Anderson DM Kumaki S Park LS Grabstein KH Cosman D Il-15, a novel T cell growth factor that shares activities and receptor components with IL-2 J Leukocyte Biol 1995 57 763 766 7759955 Armitage RJ Mcduff BM Eisenman J Paxton R Grabstein KH Il-15 has stimulatory activity for the induction of B cell proliferation and differentiation J Immunol 1995 154 483 490 7814861

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==== Front Int J Behav Nutr Phys ActThe International Journal of Behavioral Nutrition and Physical Activity1479-5868BioMed Central London 1479-5868-1-131534165610.1186/1479-5868-1-13ResearchPredictors of vigorous exercise adoption and maintenance over four years in a community sample Boutelle Kerri N [email protected] Robert W [email protected] Simone A [email protected] Division of General Pediatrics and Adolescent Health, University of Minnesota, 200 Oak St. SE, Suite 160, Minneapolis, MN 55455, USA2 Division of Epidemiology, School of Public Health, University of Minnesota, 1300 S. Second St, Suite 300, Minneapolis, MN 55455, USA2004 1 9 2004 1 13 13 4 2 2004 1 9 2004 Copyright © 2004 Boutelle et al; licensee BioMed Central Ltd.2004Boutelle et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Very little is known about the correlates of adoption and maintenance of vigorous exercise. The purpose of this study was to understand the sociodemographic correlates of exercise adoption and maintenance in a community sample. Methods 917 women and 229 men completed annual surveys as part of a community-based weight gain prevention trial over four years. Multivariate regressions evaluated predictive factors for maintenance of vigorous exercise over time in regular exercisers, and predictors of adoption of exercise in adults who were sedentary at baseline. Results Exercise maintenance at Years 2 and 3 was associated with ethnicity and exercise level at baseline, while exercise maintenance at Year 4 was associated with television watching, BMI and exercise at baseline. Exercise level at baseline was associated with exercise initiation at Year 2 and Year 3. Income level, marital status, and smoking status predicted exercise initiation at Year 4. Conclusions Predictors of vigorous exercise maintenance were more consistent than predictors of vigorous exercise initiation. Results suggest that those who adopt vigorous exercise are a heterogeneous group and intervention messages could be more broadly focused. These data also suggest that exercise maintenance interventions should continue to target low-income populations with messages regarding smoking, weight and television. Clearly further research is needed to understand the factors that contribute to exercise initiation and maintenance, and to develop effective interventions to improve levels of physical activity levels. ==== Body Background Vigorous exercise is considered one of the key-components of a healthy lifestyle and cardiovascular fitness. Higher levels of exercise are associated with lower risks of hypertension, diabetes, osteoporosis, colon cancer, coronary heart disease, depression, anxiety, and has been shown to enhance weight control [1-3]. While earlier public health recommendations targeted vigorous physical activity, current recommendations include both vigorous and moderate physical activity [4]. Current vigorous activity recommendations include "increasing the proportion of adults who engage in vigorous physical activity that promotes the development and maintenance of cardiorespiratory fitness three or more days per week for 20 or more minutes per occasion" [5]. Although the benefits of exercise are well documented, research shows that only 15–25% of adults are engaging in vigorous exercise three or more times a week for at least 20 minutes [2,6]. More than 60% of American adults are not exercising at a moderate or vigorous level frequently enough to reap the health benefits [2]. Nearly 25% of all U.S. adults report no leisure time exercise at all [6]. Numerous studies have examined predictors of exercise maintenance among participants in organized exercise programs [7]. However, data using longitudinal community based studies are sparse. Two community studies that examined the maintenance and adoption of exercise showed that maintenance of vigorous activity over one year was associated with attitudes toward exercise, exercise knowledge, female gender, and self-efficacy [8]. Over two years, vigorous physical activity maintenance was associated with self-efficacy and younger age for initially active men and with education for initially active women [9]. Adoption of moderate activity over one year was associated with health knowledge [8]. Adoption of vigorous activity over two years was associated with self-efficacy, younger age, and neighborhood environment in men, and education, self-efficacy, and friend and family support in women [9]. Although more research is being conducted on correlates of adoption and maintenance of exercise, more longitudinal research is needed among demographically diverse community samples [10]. Considering only 10–25% of community residents have been successful in adopting even short-term leisure time exercise [8,11,12] there is a need to learn more about the people in the community who adopt and continue exercising. Research on potential correlates of long-term maintenance, initiation, and change in these correlates is needed to better understand how changes in life circumstances might be associated with changes in vigorous exercise. The present study sought to cross-sectionally and prospectively examine the correlates of regular vigorous exercise in a sample of community adults over a four year period. Specifically, this study attempted to expand on the current literature by understanding the relationship between regular vigorous exercise and exercise adoption and maintenance by evaluating three comparisons in a community sample: 1) cross-sectional differences in characteristics of regular vigorous exercisers compared to non-exercisers, 2) predictive factors for maintenance of vigorous exercise over time in people who are already exercising at a high level as compared to those who do not maintain their exercise level, and 3) predictors of adoption of exercise in community adults who were sedentary at baseline as compared to those who do not adopt vigorous exercise. Methods Sample The study sample included 917 women and 229 men who completed baseline surveys as part of a 3-year community-based weight gain prevention trial (Pound of Prevention; POP). Participants were recruited using a variety of methods, including direct mail sent to university employees, and advertisements in community newspapers, health department employee newsletters and radio public service announcements. In addition, recruitment also targeted lower-income women at commercial shopping centers and at community health department clinics. Lower income women were paid $20 to enroll in the study. All participants were informed that they would be randomly assigned to either a mail-based educational program, or a no contact control group and that they would be measured once per year for a total of 4 years. This study was approved by the University of Minnesota Institutional Review Board. Measures Study participants completed questionnaires and were measured for height and weight at baseline and at three annual data collection visits (Years 2, 3 and 4) following baseline. Measures included in the present study are listed below. Vigorous exercise Planned vigorous exercise behavior was assessed using 5 questions from a self-administered version of the Physical Activity History questionnaire (PAH). The PAH is reliable and valid and has been used in several large epidemiologic studies [13]. For this study, exercise activities were limited to those that would deliver significant cardiovascular benefit. Participants rated how frequently in the past year they had engaged in one or more of the activities listed. Response choices on the PAH included; never or less than once per month; 1–3 per month; 1–2 per week; 3–4 per week; or 5 or more per week. Five questions were used in this study to represent high intensity planned exercise; 1) Vigorous jogging, running, backpacking or mountain climbing, 2) Bicycle faster than 10 MPH or exercise hard on an exercise bicycle or rowing machine, 3) Vigorous exercise class or vigorous dance, 4) Brisk walking, hiking, skating or cross-country skiing or 5) other vigorous exercise (including lap distance swimming, vigorous racket sports and other strenuous sports such as competitive basketball, football, volleyball and soccer). Hours spent watching television To evaluate competing activities, we evaluated participant's report of time spent watching television. Time spent watching television has been positively correlated with body weight, presumably in part due to its displacement of exercise behaviors [14]. Participants reported the number of hours they watch television on an average day. Social Support Social support from family and friends has been found to be related to exercise in a number of studies [9]. Participants in this study ranked both their family and friends on the extent to which they were supportive of healthy eating and exercise behaviors on a 5 point scale, with 1 representing "Not at all helpful" to 5 representing "Very helpful". Body Mass Height was measured to the nearest centimeter using a wall-mounted ruler and weight was measured to the nearest half pound using calibrated balance beam scales. Body mass index was calculated using the formula weight (kg)/height (m2). Demographic Information All demographic information was self-reported and included age, educational attainment (highest level completed), gender, employment status, income, ethnicity, smoking status and marital status. Statistical Analysis Before performing analyses evaluating the predictors of exercise maintenance and initiation, the possibility of an intervention effect was evaluated. Maintenance of high intensity exercise at Year 2 was not significantly associated with treatment, however, maintenance of high intensity exercise at Years 3 and 4 were associated with treatment status. Intervention participants were more likely to be maintaining exercise behavior at Year 3 (X2 = 14.14, p = .042) and at Year 4 (X2 = 6.42, p = .011). Initiation of exercise at Year 2 and Year 3 were not significantly associated with treatment status. However, initiation of exercise at Year 4 was marginally positively associated with treatment (X2 = 3.30, p = .069). Due to these associations, treatment assignment (intervention vs control) was controlled in multiple regression analyses. Participants were classified as regular vigorous exercisers (> 3 times per week) and non-regular exercisers (< 3 times per week) based on their responses to the five planned high-intensity exercises on the PAH at each of the 4 assessment points. Using these classifications (yes/no vigorous exercise), the participants were classified as maintainers if they exercised more than three times per week at both baseline and at a subsequent annual evaluation (Year 2, 3, or 4), and non-maintainers if they exercised three times per week at baseline and less than three times per week at a subsequent annual evaluation (Year 2, 3, or 4). Similarly, participants were classified as initiators if they reported less than three exercise sessions per week at baseline and more than three exercise sessions per week at subsequent annual evaluation (Year 2, 3, or 4), and non-initiators if they exercised less than three times per week at both baseline and a subsequent annual evaluation (Year 2, 3, or 4). Descriptive analyses using t-tests and chi-square tests were utilized to assess univariate associations between the baseline predictor variables and exercise status. Maintainers were compared with non-maintainers, and consistently sedentary participants were compared with participants who adopted vigorous activity at Years 2, 3 and 4. Logistic regression was used to further assess the statistical significance of associations between the predictor variables and exercise maintenance and adoption. These univariate logistic regression models predicted exercise maintenance (yes/no) and exercise initiation (yes/no) at Years 2, 3 and 4 using the baseline demographic factors as independent variables. Furthermore, the variables that were significant in the univariate analyses were entered into a multivariate logistic regression model to assess the relative weight and statistical significance of associations between the predictor variables and the variables representing exercise maintenance and initiation. These multivariate logistic regression models predicted exercise maintenance (yes/no) and exercise initiation (yes/no) at Years 2, 3 and 4. In these multivariate regressions, we controlled for exercise level at baseline and treatment status. All statistical analyses were conducted using the SAS Version 6.12 [15]. Since other research studies have indicated gender differences in predictors of exercise maintenance and initiation [16], the gender by exercise interaction was evaluated in a regression model using the frequency of vigorous exercise per week at Year 2 as the dependent variable. The independent variables in this model were the main effects for gender, the main effect for vigorous exercise at baseline, and the gender by vigorous exercise at baseline interaction. The interaction was not significant and thus the data were not stratified for analyses. Results Cross-sectional sample description Using the definition of vigorous exercise described above, 564 participants (453 females and 111 males) vigorously exercised more than 3 times per week and 582 participants (464 females and 118 males) did not vigorously exercise more than 3 times per week at baseline. Sociodemographic characteristics of these participants are described in Table 1. Not surprisingly, participation in vigorous exercise was associated with being employed, non-smoker, lower BMI, fewer hours spent watching television, and higher perceived social support from family and friends. None of the other demographic variables were significantly associated with vigorous exercise at baseline. Table 1 Correlates of exercise status at baseline Exercisers < 3 X/wk at baseline N = 582 Exercisers > 3 X/wk at baseline N = 564 Gender Female 464 (79.7%) 453 (80.3%) Age (years) 37.6 (SD = 6.3) 37.6 (SD = 7.2) BMI (kg/m2) 28.1 (SD = 6.3) 26.2 (SD = 5.2)*** Employed Yes 474 (81.4%) 495 (87.8%) Ethnicity White 500 (85.9%) 503 (89.1%) Income group < 25 K 218 (37.5%) 190 (33.8%) Marital Status Married 288 (49.5%) 266 (47.2%) Sep/Div/Wid 96 (16.5%) 96 (17.0%) Never Married 198 (34.0%) 202 (35.8%) Education HS Degree or less 73 (12.54%) 70 (12.4%) Some college 235 (40.4%) 192 (34.0%) College degree or more 274 (47.1%) 302 (53.6%) Smoking Yes 131 (22.5%) 80 (14.2%)*** TV hours/day 2.6 (SD = 2.5%) 2.1 (SD = 1.9%) Social support family 2.7 (SD = 1.3) 2.9 (SD = 1.3) Social support friend 2.6 (SD = 1.2) 3.0 (SD = 1.2)*** *** denotes significant difference at the p < .001 Prospective analyses evaluating predictors of exercise maintenance Prospective univariate evaluations of maintenance of exercise at the three annual evaluations Maintainers and non-maintainers were compared on baseline demographic variables, social support, hours spent watching television and smoking. Similar patterns of associations were found for annual visits at Years 2, 3 and 4. Participants who maintained their exercise level at evaluation Years 2, 3 and 4 weighed less, were employed, Caucasian, of a higher income group, more highly educated, more likely to be non-smokers and watched less television per day at baseline. At evaluation Years 3 and 4, the maintainers were older than the non-maintainers at baseline. The maintainers and non-maintainers did not report differences in marital status or social support. These results are presented in Table 2. Table 2 Associations between exercise maintenance and baseline demographic, smoking, social support and hours watching television. Year 2 Year 3 Year 4 Maintain N = 378 Non-maintain N = 186 Maintain N = 277 Non-maintain N = 230 Maintain N = 216 Non-maintain N = 256 Gender Female 300 79.4% 153 82.3% 214 77.3% 194*** 84.4% 170 78.7% 209 81.6% Age (years) 38.0 SD = 6.6 36.9 SD = 8.4 38.3 SD = 6.6 36.9** SD = 8.0 38.7 SD = 6.5 37.1** SD = 7.9 BMI (kg/m2) 25.5 SD = 4.7 27.8*** SD = 6.0 25.7 SD = 4.7 26.7** SD = 5.8 25.4 SD = 4.6 27.0*** SD = 5.8 Employed Yes 341 90.2% 154*** 82.8% 250 90.3% 197 85.7% 200 92.6% 217*** 84.8% Ethnicity White 350 92.6% 153*** 82.3% 259 93.5% 192*** 83.5% 203 94.0% 222*** 86.7% Income Group < $25,000 111 29.4% 79*** 42.5% 82 29.6% 89** 38.9% 57 26.4% 108*** 42.2% Marital Status Married 181 47.9% 85 45.7% 130 46.9% 108 47.0% 106 49.1% 115 44.9% Sep/Div/ Widowed 59 15.6% 37 19.9% 42 15.2% 39 17.0% 35 16.2% 49 19.1% Never Married 111 29.4% 79 42.5% 105 37.9% 83 36.1% 75 34.7% 92 35.9% Education HS or less 35 9.26% 35 18.8% 23 8.3% 40 17.4% 15 6.9% 41 16.0% HS + some college 128 33.8% 64 34.4% 93 33.6% 79 34.4% 73 33.8% 96 37.5% College or more 215 56.9% 87*** 46.8% 161 58.1% 111 48.3% 128 59.3% 119*** 46.5% Smoking yes 42 11.1% 38*** 20.4% 29 10.5% 39** 16.7% 24 11.1% 45** 17.6% TV/day 1.8 SD = 1.8 2.5*** SD = 2.20 1.78 SD = 1.6 2.4*** SD = 2.1 1.7 SD = 1.5 2.4*** SD = 2.1 Social support family 3.0 SD = 1.4 2.8 SD = 1.2 3.0 SD = 1.4 2.9 SD = 1.3 3.0 SD = 1.4 2.9 SD = 1.3 Social support friend 3.0 SD = 1.2 3.1 SD = 1.2 3.0 SD = 1.2 3.0 SD = 1.2 3.0 SD = 1.2 3.0 SD = 1.2 ** denotes significant difference at the p < .05 *** denotes significant difference at the p < .001 Prospective multivariate predictors of exercise maintenance Multivariate logistic regression analyses were performed to examine the predictors exercise maintenance at Years 2, 3 and 4. Demographic variables at baseline (BMI, income group, employment status, education, ethnicity, smoking status, hours watched TV/day, age, gender) were entered into logistic regression model along with exercise level at baseline and treatment group. Exercise maintenance at Years 2, 3 and 4 were the dependent variables in the three models. At Year 2, ethnicity (OR = .52, CI = .27–.99), BMI (OR = .93, CI = .89–.96) and exercise at baseline (OR = 1.21, CI = 1.11–1.31) were significant predictors of exercise maintenance. Ethnicity (OR = .463, CI = 0.229–0.933) and exercise at baseline (OR = 1.21, CI = 1.12–1.31) were significant predictors of exercise maintenance at Year 2. Television hours per day (OR = 0.86, CI = .74–.99), BMI (OR = .95, CI = .91–.99) and exercise at baseline (OR = 1.22, CI = 1.13–1.31) were significant predictors of exercise maintenance at Year 4. Prospective analyses evaluating predictors of exercise initiation Univariate evaluations of initiation of exercise at the three annual evaluations Results of univariate analyses relating exercise initiation with baseline variables are shown in Table 3. Participants who initiated a vigorous exercise program differed from consistently sedentary persons in ways that were similar to those observed for exercise maintenance. These results, however, were weaker in magnitude and not as consistent over the three time periods as the results from the exercise maintenance analyses. At baseline, initiators at Year 2 had a higher income, reported higher social support from family and friends, and had a lower BMI. The only significant difference between the initiators and the sedentary persons was hours watching television per day at Year 3. Finally, initiators at Year 4 were more likely to be married at baseline. Table 3 Associations between exercise initiation and baseline demographic, smoking, social support and hours watching television. Year 2 Year 3 Year 4 Adopt N = 135 Sedentary N = 447 Adopt N = 73 Sedentary N = 347 Adopt N = 48 Sedentary N = 326 Gender Female 108 80.0% 356 79.6% 51 69.9% 305*** 81.5% 36 75.0% 269 82.5% Age (years) 37.9 SD = 6.3 37.5 SD = 6.3 38.2 SD = 5.2 37.4 SD = 6.5 37.4 SD = .6.7 37.4 SD = 6.6 BMI (kg/m2) 27.34 SD = 6.1 28.4* SD = 6.3 27.6 SD = 5.5 28.5 SD = 6.4 28.3 SD = 6.9 28.6 SD = 6.4 Employed Yes 114 84.4% 360 80.5% 63 86.3% 297 79.4% 38 79.5% 259 79.5% Ethnicity White 118 87.4% 382 85.5% 63 86.3% 319 85.3% 42 87.5% 277 84.5% Income Group < $25,000 41 30.4% 177** 39.6% 26 35.6% 151 40.4% 20 41.7% 131 40.2% Marital Status Married 69 51.1% 219 49.0% 33 45.2% 186 49.7% 30 62.5% 156 47.9% Sep/Div/ Widowed 22 16.3% 74 16.5% 13 17.8% 61 16.3% 9 18.8% 52 16.0% Never Married 44 32.59% 154 34.5% 27 37.0% 127 34.0% 9 18.8% 18.8* 36.2% Education HS degree or less 14 10.4% 59 13.2% 7 9.6% 52 13.9% 5 10.4% 47 14.4% HS degree + some college 49 36.3% 186 41.6% 25 34.3% 161 43.1% 19 39.6% 142 43.6% College degree + more 72 53.3% 202 45.2% 41 56.2% 161 43.1% 24 50.0% 137 42.0% Smoking status yes 25 18.5% 106 23.7% 15 20.6% 91 24.3% 6 12.5% 85** 26.1% TV/day 2.4 SD = 1.9 2.7 SD = 2.6 2.1 SD = 1.6 2.8*** SD = 2.8 2.6 SD = 3.2 2.8 SD = 2.7 Social support family 3.0 SD = 1.3 2.7*** SD = 1.3 2.6 SD = 1.4 2.7 SD = 1.3 2.9 SD = 1.2 2.6 SD = 1.3 Social support friend 2.8 SD = 1.2 2.6** SD = 1.2 2.5 SD = 1.1 2.6 SD = 1.2 2.5 SD = 1.2 2.6 SD = 1.2 *** denotes significant difference at the p < .001 ** denotes significant difference at the p < .05 Prospective multivariate predictors of exercise initiation Logistic regression analyses were also performed to examine the predictors of exercise initiation and Years 2, 3 and 4. Similar to the evaluations for exercise maintenance, demographic variables at baseline which were significant in the univariate analyses (baseline BMI, income group, marital status, gender, smoking, hours spent watching television, family social support and friend social support) were entered into three separate models controlling for exercise level at baseline and treatment group to identify predictors exercise initiation at Years 2, 3 and 4. Four variables were significant predictors of exercise initiation at one or more time points. Exercise level at baseline was positively related to exercise initiation at Year 2 (OR = .23, CI = 1.71–2.90) and at Year 3 (OR = 1.71, CI = 1.23–2.38). Baseline income group (OR = 2.38, CI = 1.08–5.23), marital status (OR = 6.07, CI = 0.39–0.95) and smoking status (OR = 3.53, CI = 0.14–0.91) predicted exercise initiation at Year 4. Discussion This study evaluated the demographic predictors of vigorous exercise initiation and maintenance in a community sample. To our knowledge, this is the first study to look at baseline demographic factors as predictors for exercise adoption and maintenance over a four-year period. This study found that demographic predictors were more consistent for exercise maintenance than exercise initiation over the four years evaluated in this study. Results showed that compared to those who did not maintain vigorous exercise, participants who maintained exercise over a 2, 3 and 4 year period were more likely to be employed, Caucasian, have a higher income, have more education, be a nonsmoker, watch less television and have a lower BMI. One of the interesting findings was that correlates of exercise initiation were less consistent than correlates of exercise maintenance over time. There did not appear to be a consistent pattern that described the associations between the predictors included in this study and vigorous exercise initiation. Although certain predictors were associated with exercise initiation at Years 2, 3 or 4, such as gender or BMI, none of the predictors were consistent over the years measured in the study. These results suggest that people in community populations who initiate vigorous exercise are a heterogeneous group, and there may be greater difficulties predicting who will initiate exercise. It could be much simpler to identify the participants who will be more likely to continue exercise, as compared to the participants who will adopt exercise. It is possible that changes in attitudes, life circumstances (such as sickness), new relationships, or variables that were not measured in this study may be better predictors of exercise adoption or maintenance. However, since we did not measure these variables, we can only speculate whether they may disrupt exercise patterns. The processes of vigorous exercise adoption may be better represented by theoretical understandings of exercise behavior, such as the health belief model [17], transtheoretical model [18,19], social cognitive theory [20], or the theory of planned behavior[21]. These results may have implications for designing and implementing exercise interventions. The results support others studies that suggest targeting exercise maintenance interventions at the lower income participants. However, to achieve the Healthy People 2010 objectives by increasing participation rate in vigorous exercise, interventions need to be designed to promote exercise adoption. Unfortunately, the present study's results only suggest that we can not characterize this group well. This lack of characterization of this group may actually be a benefit for interventions. Ethnic group, gender, and socio-economic status did not predict exercise initiation in this study. This suggests that each group was as likely to begin a vigorous exercise program. This is good news as exercise initiation may not be limited to those who can afford health clubs and trainers, or those who are the member of a specific ethnic group or gender. Although previous studies have stratified participants by gender [16], no interaction was found in this study between gender and exercise status. The only associations with gender were seen during Year 3, when fewer women than men maintained exercise and fewer women than men initiated exercise. It is possible that the gender by exercise interaction was not significant due to the limitation of exercise in the vigorous activity range, versus the moderate or mild activity range. One hypothesis is that the predictors of exercise adoption that are gender specific may have to do with moderate or minimal exercise adoption. Strengths and limitations of the present study should be recognized. This is one of a few studies that evaluate predictors of exercise initiation, and the first to evaluate exercise initiation over a four-year period. In addition, the sample is large, diverse, and includes longitudinal measurements. This study did not evaluate the predictors of moderate exercise initiation or maintenance. Thus, these results can not be directly interpreted for public health interventions targeted at increasing moderate activity in the general population. In addition, this study may also be affected by a selection bias. The participants in this study volunteered for a study on weight gain prevention, for which the participants may have expected to include a message on increasing exercise. Of note, 49% of the sample reported vigorous exercise more than 3 times per week at baseline, which is much higher than the 15–25% reported in national surveillance studies [6,2]. Although treatment status was controlled for in the analyses, these participants were recruited for an intervention trial, rather than a cohort trial, which could also contribute to a selection bias. In addition, the measures in this study are self-report and include few theoretically based variables. It is possible that the self-report nature of exercise in this study may have allowed over-reporting of vigorous exercise. Considering these limitations, this study does add to the knowledge base about who initiates and maintains vigorous exercise. The results suggest that vigorous exercise maintenance interventions should continue to target low-income populations and that interventions could incorporate messages regarding smoking, weight control, and television. We found that those who adopt vigorous exercise are a more heterogeneous group, and that no one group is more likely to adopt exercise than the others. This suggests that vigorous exercise intervention messages could be more broadly based. This study also suggests that further research is needed to identify participants and effective interventions for those who begin exercise programs. Competing interests None declared. Authors contributions KB conceptualized the study, planned and executed the analyses, interpreted the results, and drafted the manuscript. RJ participated in conceptualization of the study, interpretation of the results and assisted in drafting the manuscript. SF participated in conceptualization of the study, interpretation of the results and assisted in drafting the manuscript. All authors read and approved the final manuscript. ==== Refs Powell KE Thompson PD Caspersen CJ Kendrick JS Physical activity and the incidence of coronary heart disease Annu Rev Public Health 1987 8 253 287 3555525 10.1146/annurev.pu.08.050187.001345 U.S. Department of Health and Human Services Physical activity and health: A report of the surgeon general In Atlanta, GA: Centers of Disease Control and Prevention, National Center for Chronic Disease Prevention and Health Promotion 1996 Blair SN Jacobs DR JrPowell KE Relationships between exercise or physical activity and other health behaviors Public Health Rep 1985 100 172 180 3920715 Pate RR Pratt M Blair SN Haskell WL Macera CA Bouchard C Buchner D Ettinger W Heath GW King AC Physical activity and public health. A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine Jama 1995 273 402 407 7823386 10.1001/jama.273.5.402 U.S. Department of Health and Human Services Healthy People 2010: Understanding and Improving Health In Washington, DC 2000 2 U.S.: Government Printing Office National Center for Health Statistics Healthy People 2000; Final Review In Hyattsville, MD: Public Health Service 2001 Dishman RK Dishman RK Overview Exercise adherence: It's impact on public health 1988 Champaign, IL: Human Kinetics Press Sallis JF Haskell WL Fortmann SP Vranizan KM Taylor CB Solomon DS Predictors of adoption and maintenance of physical activity in a community sample Prev Med 1986 15 331 341 3763558 10.1016/0091-7435(86)90001-0 Sallis JF Hovell MF Hofstetter CR Barrington E Explanation of vigorous physical activity during two years using social learning variables Soc Sci Med 1992 34 25 32 1738853 10.1016/0277-9536(92)90063-V Marcus BH Dubbert PM Forsyth LH McKenzie TL Stone EJ Dunn AL Blair SN Physical activity behavior change: issues in adoption and maintenance Health Psychol 2000 19 32 41 10709946 Young DR Haskell WL Jatulis DE Fortmann SP Associations between changes in physical activity and risk factors for coronary heart disease in a community-based sample of men and women: the Stanford Five-City Project Am J Epidemiol 1993 138 205 216 8356962 Eaton CB Reynes J Assaf AR Feldman H Lasater T Carleton RA Predicting physical activity change in men and women in two New England communities Am J Prev Med 1993 9 209 219 8398220 Jacobs DR Hahn LP Haskell WL Pirie P Sidney S Validity and reliability of short physical activity hisotry: Cardia and the Minnesota Heart Health Program Journal of Cardiopulmonary Rehabilitation 1989 9 448 459 Jeffery RW French SA Preventing weight gain in adults: design, methods and one year results from the Pound of Prevention study Int J Obes Relat Metab Disord 1997 21 457 464 9192229 10.1038/sj.ijo.0800431 SAS Institute SAS/STAT Software: changes and enhancements through release 6.12 Cary, NC: SAS Institute 1997 Sallis JF Hovell MF Hofstetter CR Predictors of adoption and maintenance of vigorous physical activity in men and women Prev Med 1992 21 237 251 1579558 10.1016/0091-7435(92)90022-A Rosenstock IM Strecher VJ Becker MH Social learning theory and the Health Belief Model Health Educ Q 1988 15 175 183 3378902 Prochaska JO DeClemente CS Transtheoretical therapy: toward a more integrative model of change Psychotherapy Theory Research Practice 1982 20 161 173 Prochaska JO Velicer WF The transtheoretical model of health behavior change Am J Health Promot 1997 12 38 48 10170434 Bandura A Social foundations of thought and action: a social-cognitive theory 1986 Englewood Cliffs, NJ: Prentice-Hall Ajzen I Kuhl J, Beckman J From intentions to actions: a teory of planned behavior Action-control: from cognition to behavior 1985 New York: Springer 11 39

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1161533314610.1186/1471-2105-5-116Methodology ArticleApplying Support Vector Machines for Gene ontology based gene function prediction Vinayagam Arunachalam [email protected]önig Rainer [email protected] Jutta [email protected] Falk [email protected] Roland [email protected] Karl-Heinz [email protected] Sándor [email protected] Department of Molecular Biophysics, Deutsches Krebsforschungszentrum (DKFZ), TP3, Im Neuenheimer Feld 580, Heidelberg, D-69120, Germany2 Theoretical Bioinformatics, Deutsches Krebsforschungszentrum (DKFZ), TP3, Im Neuenheimer Feld 580, Heidelberg, D-69120, Germany3 Institut für Medizinische Biometrie, Epidemiologie und Informatik (IMBEI), Johannes Gutenberg-Universität Mainz, 55101, Mainz, Germany2004 26 8 2004 5 116 116 11 5 2004 26 8 2004 Copyright © 2004 Vinayagam et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The current progress in sequencing projects calls for rapid, reliable and accurate function assignments of gene products. A variety of methods has been designed to annotate sequences on a large scale. However, these methods can either only be applied for specific subsets, or their results are not formalised, or they do not provide precise confidence estimates for their predictions. Results We have developed a large-scale annotation system that tackles all of these shortcomings. In our approach, annotation was provided through Gene Ontology terms by applying multiple Support Vector Machines (SVM) for the classification of correct and false predictions. The general performance of the system was benchmarked with a large dataset. An organism-wise cross-validation was performed to define confidence estimates, resulting in an average precision of 80% for 74% of all test sequences. The validation results show that the prediction performance was organism-independent and could reproduce the annotation of other automated systems as well as high-quality manual annotations. We applied our trained classification system to Xenopus laevis sequences, yielding functional annotation for more than half of the known expressed genome. Compared to the currently available annotation, we provided more than twice the number of contigs with good quality annotation, and additionally we assigned a confidence value to each predicted GO term. Conclusions We present a complete automated annotation system that overcomes many of the usual problems by applying a controlled vocabulary of Gene Ontology and an established classification method on large and well-described sequence data sets. In a case study, the function for Xenopus laevis contig sequences was predicted and the results are publicly available at . ==== Body Background Ongoing genome sequencing and recent developments in cDNA sequencing projects have led to an exponential rise in the amount of sequence information. This has increased the need for acquiring knowledge from sequences as to their biological function. Annotating a single sequence is the gateway to interpreting its biological relevance. However, the usefulness of these annotations is highly correlated with their quality. Accurate annotation has traditionally been maintained manually with the experience of individual experts and the experimental characterisation of sequences. However, the increasing gap between the amount of sequence data available and the time needed for their experimental characterisation demands computational function prediction in complementing manual curation [1-4]. Commonly, computational functional assignment is based on homologues identified from database searches [5]. Such an automated annotation process provides comparable results due to a uniform analysis of all query sequences across the same databases and the possibility of repeating the annotation to updated sequence data [6]. However, crucial aspects for consideration in automated annotation are i) the problems associated with the databases themselves: sequence errors, erroneous annotation due to spelling ambiguities, incomplete functional annotation, inconsistent functional annotation across databases, consistent but wrong annotation across databases, and ii) the problems associated with the inference, i.e. false positives, where an assignment is made on the basis of a wrongly inferred homology [3,7,8]. A number of excellent annotation systems have been developed to tackle these problems, e.g. RiceGAAS [9], GAIA [10], Genotator [11], Magpie [12], GeneQuiz [6], GeneAtlas [13] and PEDANT [14]. However, little has been done to quantify the annotation accuracy by defined benchmarks and establish a method to provide a confidence value for each annotation. The current annotation, written in a rich, non-formalised language also complicates this automated process. We addressed this problem by applying a controlled vocabulary from Gene Ontology (GO) [15-17]. GO provides consistent descriptions of gene products in a species-independent manner. The GO terms are organised in structured, controlled vocabularies (ontologies) to describe gene products in terms of their associated biological processes, cellular components and molecular functions. An increasing number of GO-mapped sequence databases make it possible to replace traditional database searches with GO-related searches. These include databases such as GenBank [18], SWISS-PROT [18], SwissPROT/TrEMBL [19], the TIGR Gene Index [20] and several other genome databases. Many annotation approaches have now been developed based on Gene Ontology. The uncharacterised sequences are searched across GO-mapped protein databases and assigned with GO terms of the best hits [21,22]. Jensen and co-workers used neural networks to predict specific subsets of GO terms [23]. Furthermore, Schung et al predicted GO terms by intersecting domain profiles [24]. The SwissPROT/TrEMBL entries were associated with GO terms by an automated process coupled with manual verification [19]. Text mining and similarity searches were combined to annotate SWISS-PROT and GenBank entries with GO terms [18]. However, these approaches were either applied to specific GO subsets or did not provide defined benchmarks and confidence values for their predictions. We have developed an automated system for large-scale cDNA function assignment, designed and optimised to achieve a high-level of prediction accuracy without any manual refinement. Our system assigns molecular function GO terms to uncharacterised cDNA sequences and defines a confidence value for each prediction. The cDNA sequences were searched against GO-mapped protein databases and the GO terms were extracted from the homologues. In the training phase, these GO terms were compared to the GO annotation of the query sequences and labelled correspondingly. We applied Support Vector Machines (SVMs) as the machine learning method to classify whether the extracted GO terms were appropriate to the cDNA sequence or not. In order to classify the GO terms we used a broad variety of elaborated features (attributes) including sequence similarity measures, GO term frequency, GO term relationships between homologues, annotation quality of the homologues, and the level of annotation within the GO hierarchy. To enhance the reliability of the prediction, we used multiple SVMs for classification and applied a committee approach to combine the results with a voting scheme [25]. The confidence values for the predicted GO terms were assigned based on the number of votes i.e. number of SVMs predicting particular GO term as correct. The performance of the system was benchmarked with 36,771 GO-annotated cDNA sequences derived from 13 organisms. It achieved 80% precision for 74% of the test sequences. We applied our annotation system to predict the function for Xenopus laevis, a widely studied model organism in developmental biology. Because many researchers are now focussing on the functional genomics of this organism, a demand exists for a quality annotation [26]. Therefore we applied our system to improve the quality and coverage of the existing annotation. We predicted the function for 17,804 Xenopus laevis contig sequences (from TIGR Gene Indices) yielding annotation with good confidence values for more than half of these sequences. Results General workflow of training and classification The classifier (SVM) needs to specify attribute values (features) for a broad list of samples and a class label for each of these samples. Through the training samples it learns the feature patterns and tries to group them according to their class labels. After training, the algorithm assigns class labels to new samples according to the class that they best match. We selected GO-annotated cDNA sequences for training the SVM classifier. The nucleotide sequences were searched against GO-mapped protein databases and GO-annotations were extracted from the significant hits. Then, each GO term obtained was utilized as a sample for the feature table. The sample GO terms were then labelled as either correct ("+1") or false ("-1") by comparing them to the original annotation. Note that we applied the relationships of the GO terms based on their graph structure: "Correct" was assigned not only if they were exact matches but also if the GO terms were related as either "parent" or "child" (Figure 1). Next, the samples were attached with their features or attributes, calculated from the BLAST [27] results. With this data, the classifier was trained to distinguish between the attribute patterns that contributed to class +1 (correct prediction of a GO term) and class-1 (false prediction). To predict the function of unknown sequences, the same procedure was applied as for the training sequences in order to obtain their GO terms and corresponding attribute values. According to these attribute values, the classifier assigned a class for every GO term of the BLAST hits (Figure 2). Datasets for training and testing SVM For training and testing the SVM, we selected 39,740 GO-annotated cDNA sequences from the following organisms: Saccharomyces cerevisiae (yeast), Drosophila melanogaster (fly), Mus musculus (mouse), Arabidopsis thaliana (Arabidopsis), Caenorhabditis elegans (worm), Rattus norvegicus (rat), Danio rerio (fish), Leishmania major (Leishmania), Bacillus anthracis Ame (Bacillus), Coxiella burnetii RSA 493 (Coxiella), Shewanella oneidensis MR-1 (Shewanella), Vibrio cholerae (Vibrio) and Plasmodium falciparum (Plasmodium) (Table 1). From these, 55.3% of the cDNA sequences were contributed by Arabidopsis, mouse and fly (22.1%, 18%, and 15.2% respectively). Prokaryotic bacteria (Bacillus, Coxiella, Shewanella and Vibrio) contributed 20.6% and the remaining 24.1% of the sequences came from rat, fish, worm, Plasmodium, Leishmania and yeast. Yeast and fly are purely manually annotated datasets. Where as Bacillus, Coxiella, Vibrio, Shewanella, Leishmania and Plasmodium are mostly manually, and the rest mostly automatically annotated datasets. Manual annotation tends to be conservative and sparse, since the GO terms are assigned only if the annotator is highly confident. Therefore, a GO term may be missed due to a poor definition of a false negative. To reduce this critical problems, yeast and fly annotations are accompanied by an "unknown molecular function" term for sequences with questionable further functions. To reduce false negatives, we discarded all sequences with these tags for training and testing (yeast: 2999 discarded out of 6355, fly: 8495 out of 14335). The cDNA sequences were searched across the protein databases covering a wide range of organisms from prokaryotes to eukaryotes and SWISSPROT. For 36,771 sequences we got hits with GO terms, contributing to 856,632 sample GO terms and yielding an average of 23.29 GO terms per query sequence (Table 1). These 856,632 samples were used to train our classifier. Generally, the number of GO terms per sequence was less for prokaryotes than for eukaryotes. Rat had the maximum number of GO terms per sequence (36.9), followed by fish (32.1) and worm (27.13). In contrast, Shewanella, Coxiella and Vibrio sequences had the lowest number of GO terms per sequence (10.78, 12.33 and 12.54, respectively). SVM training and testing SVM training We set up multiple classifiers by splitting the whole dataset (856,632 samples) into 99 equal subsets. Note that, amongst these 99 subsets, 96 contained data from a single organism and the remaining 3 from two organisms each. Subsequently, we built 99 classifiers with these subsets. Since the training sets were created organism-wise, the classifiers were trained from different ranges of data, based on purely manual annotation (yeast, fly), mostly automated annotation or a mixture of both. For training each of these classifiers, we performed a model selection (parameter optimisation by cross-validation; see Methods), which yielded varying accuracy values ranging from 78.81% to 96.03%, with an average accuracy of 85.11%. SVM testing To test the classifiers performance, we prepared 13 test sets (each set corresponding to a single organism) using the same 856,632 sample GO terms. The prediction quality of all 99 classifiers were assessed by an organism-wise cross-validation approach, i.e. for each organism (test set), we used all the classifiers for prediction except those that corresponded to the same organism. With this approach, we were able to simulate the annotation of a new organism. The number of classifiers used for predictions varied highly across organisms (maximum: Plasmodium and Leishmania, 98 classifiers; minimum: Arabidopsis, 74 classifiers). The quality of the predictions was estimated by comparing the predicted terms with the original annotation and the results were expressed in terms of precision and accuracy values (see Methods). The average-accuracy refers to the average of the accuracy values attained by all classifiers used for the prediction. The maximum average-accuracy was achieved for fly (81.51%), followed by yeast (80.50%), and the minimum for mouse (76.0%). Additionally, we compared the classification efficiency of the classifier derived from automatic annotation (mouse, worm and Arabidopsis) with the manually annotated test sequences (yeast and fly). The prediction of the yeast and fly sequences with the 20 classifiers from the mouse sequences produced an average-accuracy of 79% and 80% respectively. Similar results were acquired with the 25 classifiers from Arabidopsis (79% and 80%). Likewise, the worm classifiers (11 classifiers) yielded the average-accuracy of 82% for yeast and 83% for fly. These values were comparable with the average-accuracy of 81% achieved by both, using yeast as test sequences against fly classifiers (16 classifier) and vice-versa (fly test sequences against yeast classifiers). Likewise, we classified the mouse test sequences against yeast classifiers (5 classifier) and fly classifiers yielding 69% and 71% average-accuracy respectively. Combining multiple classification results by the committee approach Though we already achieved a good accuracy with some of the classifiers, our intention was to improve the precision and, furthermore, to obtain confidence values for the predicted GO terms. To this end, we combined the predictions of multiple classifiers by the committee approach. If a classifier predicted a particular GO term as correct, it contributed a vote. Votes were collected from all classifiers and summed up to yield a final score value. If no vote supported a GO term as correct, it was assigned with the label "false". Otherwise, the number of votes provided a measure of the reliability. Figure 3 shows precision and accuracy versus the number of votes. If we made predictions with a minimum of one vote, we were able to achieve 43% precision and 59% accuracy. When the stringency was raised to 25 votes, a minimum of 25 votes was required to classify a GO term as correct, yielding an accuracy of 84% and precision of 75%. At a cut-off value of 74 votes, we attained 91% precision and 71% accuracy. A cut-off value of 94 votes gave 100% precision and 67% accuracy. Our accuracy reached a plateau at 20 votes. However, it decreased slightly for stringencies of more than 30 votes. Note, that this was due to the increasing number of false negatives. The relation between the precision and the number of votes (Figure 3) was used as a means of calibrating to assign the confidence values for new predictions. For each threshold value of the votes, we calculated the sensitivity and the false positive rate to obtain a Receiver Operating Characteristic plot (ROC; Figure 4). The graph shows that the classification performance was comparable for different classes of organisms like prokaryotes, single cell eukaryotes and multi-cellular eukaryotes, which reflect the organism-independent performance of our method. Note that for fish, worm, Plasmodium and Leishmania the classification performance was particularly good due to the low number but well characterised test sequences. We compared the prediction performance for GO terms annotated with the evidence code IEA (automated annotation) and non-IEA (manually verified annotation). All sequences from Bacillus, Coxiella, Vibrio, Shewanella, yeast, Leishmania, and Plasmodium were non-IEA annotated and 99.5% of the fly GO terms were non-IEA annotated. In contrast, all sequences from fish and worm were IEA annotated. The remaining test organisms were mostly IEA annotated (rat: 88%, Arabidopsis: 79.4%, and mouse: 69.5%). The classification performances revealed by the ROC plots were comparable between IEA and non-IEA annotated test organisms (Figure 4). Therefore, the classifier could reproduce the annotation of other automated systems as well as high-quality manual annotation. We were interested in the coverage of sequences with respect to the average precision of the annotations (shown in Figure 5). Considering 1 vote as the cut-off value, we obtained 52% average precision for 98% coverage. We obtained 80% average precision for 74% coverage (cut-off: 34 votes), and 90% average precision for 42% coverage (cut-off: 65 votes). These coverage values varied when regarding the test organisms individually. The coverage for different test organisms at 80% average precision were: fish 97%, Coxiella 89%, worm 88%, Vibrio 86%, rat 85%, Bacillus 83%, Plasmodium 81%, mouse 78%, Leishmania 76%, Shewanella 74%, Arabidopsis 69%, fly 66% and yeast 57%. Xenopus annotation We extracted all Xenopus laevis contig sequences from the TIGR Xenopus laevis Gene Index (XGI) [28] and got a total of 35,251 contig sequences, excluding singletons. We applied our method to predict functional GO terms for these contig sequences. We predicted the function for 17,804 sequences with an average of 12.16 GO terms per sequence. In total, 23.4% of all the GO terms were predicted with less than 50% confidence value, 51.5% of them were between 50% to 80% confidence and the remaining 25% with a predicted confidence value of above 80%. At 80% stringency (predicted if the GO term possessed a confidence value of 80% or more), we made predictions for 9,510 contig sequences including 55,994 GO terms, yielding on average 5.88 GO terms per sequence. To compare the functional abundance of the expressed genome across the organisms, we mapped the predicted GO terms (with at least one vote) to the high-level, i.e. more generalised or high-level terms of the molecular function ontology ("GO slim" for molecular function) [29]. These molecular function GO slim nodes were taken from the second level of the molecular function ontology. The distribution of higher-level GO terms were compared between Xenopus, fly, yeast and mouse (Figure 6). Note that some of the deeper-level terms had multiple paths. They were mapped to two or more higher-level nodes, so that the total sum of the higher-level nodes exceeded 100%. Comparison to the TIGR Xenopus annotation TIGR provides a GO mapping for Xenopus contigs (TIGR Xenopus laevis gene indices). We compared our annotation with the TIGR GO annotation for molecular function. From 35,251 contig sequences, TIGR annotated 5,444 contigs with a total of 16,432 molecular function GO terms. In contrast, our approach was able to predict function terms for 17,804 contigs, i.e. more than three times that of TIGR sequences. Our procedure did not annotate 295 contigs from the TIGR annotated contigs. For the remaining 5,149 contigs, 85% of all TIGR terms were found to be exact with those using our method; 3.2% of the TIGR terms were at a higher-level of the GO tree than our annotation, so in this case we provided annotation at a deeper level; in 0.9% of the cases our annotation was at a higher-level; 8.3% of the cases were completely different; and 0.6% of the TIGR terms were obsolete. We compared the quality of TIGR and that of our annotations by a raising stringency and found that when we applied a confidence threshold of 80% for our annotation, we lost 46.6% of the sequences. This included 1,492 sequences holding equivalent TIGR annotation or 27.4% of the total TIGR annotation. With this stringency, our system annotated 9,510 contig sequences, i.e. twice the TIGR annotation at this quality. We were interested in novel annotated sequences with the highest confidence values and found we could predict GO terms for 557 contigs with a confidence value of 100% (all votes matched). Interestingly, 192 of these lacked any GO annotation by TIGR. Out of these, 184 had got a descriptive TIGR annotation and the rest had not got any. Table 2 shows the novel annotation for these eight sequences. Our novel predictions are as follows: 1) TC212171 and TC196381 are predicted to display endopeptidase activity and more specifically serine-type peptidase activity (98% and 97% confidence respectively). 2) TC209487 and TC190605 are predicted to be aminopeptidases, however for the latter the more specific prediction of prolyl aminopeptidase activity is assigned with 86% confidence. 3) TC199713 is predicted as glutathione peroxidase at 100% confidence and TC194305 is annotated as protein kinase with the same confidence. 4) Both TC187949 and TC210151 are transmembrane receptors but the latter one is classified as frizzled receptor with 82% confidence. In most of these examples the functional assignment and associated confidence were recorded in multiple levels of granularity. Discussion In this paper, we presented an automatic annotation system that is able to cope with the expanding amount of biological sequence data. Our approach efficiently combines the ongoing efforts of Gene Ontology and the availability of GO-mapped sequences with a profound machine learning system. The GO-mapped databases provide annotation described in a controlled vocabulary and also a measure of reliability, as these GO entries are labelled with their type of origin. Furthermore, GO terms are structured hierarchically, which allow us a twofold use of the information: i) the level within the tree is taken as a classification criterion to distinguish low from high-level annotations during the learning procedure, and, ii) the hierarchical structure allows us to extend hits by slightly moving up and down within a restricted local area of the tree. This may overcome fluctuations of the annotation levels coming from varying annotation experts. Our annotation system exploits the different combinations of attributes and yields functional transitivity: SVM learning and prediction are organism-independent and comparable to manual annotation, which may be supported by the nature of the attributes we utilise. Subsets and overlaps are counted in a balanced fashion to avoid biases due to the complexity of an organism and a potentially correlated complexity of its sequences. The committee approach allows us to improve the prediction quality as well as to assign confidence values for the new predictions in a straightforward manner. Our classifiers performance is hardly limited by the varying quality of the training data, whether manual or automatic annotated. The prediction results of manually annotated test sets with the classifiers based on automated annotation as well as classifiers based on manual annotation were comparable. Regarding the outcome of the overall classifiers, we achieve consistency with existing annotation from automatic annotations. This is the less complex part of our work and shows a comparable efficiency of our system. Additionally, our system reproduces annotation of purely manually annotated datasets (fly, yeast, etc). However, the performance results for these datasets are low in terms of recall, i.e. 47.4% recall with 80% precision compared to 60.6% recall with the same precision of the complete test set. Note that manual annotation tends to be conservative and sparse, yielding stringent true positive definitions, whereas automatically annotated sequences may accumulate information to a greater extent. We were interested in annotating Xenopus since it is a familiar model organism. However, the sequences were not very well annotated. Our system was applied to annotate the Xenopus contig sequences from TIGR. Through our approach, we annotated 50.5% of all contig sequences available at present, and associated a confidence value for each prediction, yielding roughly three times more sequences as compared to the currently available GO annotation. However, the coverage of annotation to new organism like Xenopus is crucial. We were able to attain predictions for 50.5% of all Xenopus contig sequences (no singletons). This compares to the applied databases that contained 53% satisfactory annotation for their sequences (not regarding sequences with unknown function terms), and better than the organism specific databases (36%). Obviously, improving the quality and quantity of annotation within the available databases goes along with the coverage exploit of machine learning algorithms for new organisms. In future we want to extend our method with the information from other sources such as domain databases and protein family databases. Conclusions We developed an automated annotation system to assign functional GO terms to an unknown sequence. We used the well-established technique of Support Vector Machines (SVM) for the classification of correct and incorrect GO terms. Our approach benefited from the broad variety of potential attributes used for the functional transitivity and a vast amount of data used for training and validating. The committee scheme exploited in our system provided a means to assign confidence values in a straightforward manner. Our system performance was robust, organism-independent and reproduced the high-quality manual annotation. When applying it to Xenopus laevis contig sequences, we obtained a remarkably enhanced annotation coverage compared to the existing annotation. Methods Quality criteria for assessing the performance of the classifier We used the following statistical terms [30,31]. Accuracy was the rate of correct predictions compared to all predictions, Accuracy: = (TP + TN) / (TP + FP + TN + FN),     (1) where TP denotes true positives, FP false positives, TN true negatives and FN false negatives. Precision was the portion of true positives with respect to all positives, Precision: = TP / (TP + FP).     (2) Also used were sensitivity := TP / (TP + FN), specificity := TN / (FP + TN), and false positive rate := 1 - specificity. We defined the term "coverage-of-sequences" as the portion of query sequences for which the classifier delivers a prediction; "Precision-per-sequence" the (average) portion of correct GO terms for a single query sequence, with respect to all GO terms assigned to it. Note that these terms were defined within our model, i.e. a good "accuracy" meant good consistency with respect to our training and test sets. Defining the GO term relationships We focused on the molecular function terms from GO, because the information extracted from the gene products is usually more predictive for determining molecular functions than for biological processes or cellular components. The functional terms and their hierarchy were obtained from the web pages of the Gene Ontology Consortium [29] (version of June 2003). In our study, relationships "is-a" and "part-of" were not distinguished. Note, that the "part-of" relationship is rare in the molecular function ontology (26 out of 6521 child-parent relationships). The annotation level varies across databases depending on the curator's individual knowledge about the gene product. To consider varying levels of annotation in the databases for similar gene products, we traced the relationships to match GO terms of different granularity for the same function. To find a relationship between two terms, the whole path of a GO term was traced back to the root (the root is the "molecular function" node, GO:0003674). We defined the distance between two GO terms as the distance of the shortest path. GO terms are organised in directed acyclic graphs, i.e. a child (more specialised term) may have multiple parents (less specialised terms). Therefore, we defined single path and multiple path relationships. In the case of single path relationships, GO terms had only one possible path to the root. The relationship of the term GO1 with respect to GO2 was classified as "parent", "child", "sibling" or "different" (Figure 1) according to the following rules: GO2 is a "parent" of GO1 if their respective paths P2 and P1 intersect in such a manner that P1 ⊂ P2,     (3) Pi denotes the set of nodes from GOi to the root GO2 is a "child" of GO1 if their paths P2 and P1 intersect such that P1 ⊃ P2,     (4) GO2 is a "sibling" of GO1 if a common parent exists with a distance of one to GO1 and GO2 (Figure 1E). To avoid ambiguities for less differentiated terms, the sibling relationship was set only, if GO1 and GO2 were at least 5 nodes away from the root. The relationship "different" was set if none of the previously stated criteria was fulfilled. We could apply the single path relationship for most of the GO terms (3665 out of 5391). However, for the remaining 1726 terms more than one path to the root were found. For these cases we defined multiple path relationships and each path was considered individually. The single path relationship was applied to each possible pair of these paths (path for GO1 and GO2, respectively) and is henceforth referred to as "path-pairs". This method could yield a list of several relations. To select the appropriate relation from this list, we considered the parent relationship to be most relevant, followed by the child relationship, and the sibling was considered least relevant. We implemented the following order: 1. The parent relationship was set if at least one of the path-pairs gave a (single path) parent relationship; 2. The child relationship was set if at least one of the path-pairs gave a child relationship. To avoid a bias due to an overwhelming number of path-pairs that did not match, we set a threshold: we considered this relationship only, if the number of path-pairs with no child relationship was equal or less than four times the number of path-pairs with child relationship; 3. The sibling relationship was set if at least one of the path-pairs gave a sibling relationship. We again set a threshold: we considered this relationship only, if the number of path-pairs with no sibling relationship was equal or less than twice the number of pairs with sibling relationship; 4. If none of these criteria could be applied, the relationship "different" was set. Note that we also implemented the hierarchy of these relations by tuning the stringencies for the fractions of path-pairs that must match (parent: no threshold, child: 1/4, sibling: 1/2). Data basis used for this study Since the function transitivity at the protein level is more reliable, we used GO-mapped protein databases for searching homologues. Gene association files were obtained via the Gene Ontology Consortium. By combining the gene association files with corresponding sequence databases we created the unified protein databases. The following organisms were used: yeast, fly, mouse, Arabidopsis, worm, rat, fish, Leishmania, Bacillus, Coxiella, Shewanella, Vibrio, Plasmodium, Oryza sativa, Trypanosoma brucei, and Homo sapiens. Apart from this, the SWISS-PROT database was also included [32]. For SVM training and testing we selected 39,740 cDNA sequences from 13 organisms. These cDNA sequences were collected from the following organisms: yeast, fly, mouse, Arabidopsis, worm, rat, fish, Leishmania, Bacillus, Coxiella, Shewanella, Vibrio and Plasmodium (see Table 1). Out of all the known cDNA sequences we extracted 39,740 with GO molecular function terms, discarding incompletely annotated ones, i.e. sequences assigned with the GO term "molecular function unknown" (GO:0005554). Computing the attributes Each cDNA sequence was searched across the protein databases, using BLASTX within the HUSAR system [33]. A query sequence was not searched within the database of their own organism. In case of SWISSPROT, hits corresponding to the query (cDNA) organism were filtered out. The BLAST files were parsed using the BLAST parser modules from W3H [34] and a low-stringent e-value cut-off of 0.01 was applied to yield a high number of possible hits. Multiple high scoring segment pairs were combined as described elsewhere [35] and used for computing the alignment features. GO terms for each database hit were extracted by considering only GO terms corresponding to the molecular function and by discarding GO terms that were prefixed with NOT (annotators state that a particular gene product is NOT associated with a particular GO term), or corresponding to "molecular function unknown" (GO:0005554). These steps reduced our dataset to 36,771 sequences, contributing to 856,632 samples. Each GO term that occurred in the hits represented a sample entry in the feature table. Below it will be referred to as "sample GO term". If a GO term occurred several times in the hits, it was considered only once. We defined 31 attributes for each GO term, representing 5 major classes of information (A)- E)): A) GO level and path: The GO structure was exploited to derive the first two attributes, A.1.GO level: the distance of the sample GO term to the root (molecular function node); A.2. GO path: the number of paths from the sample GO term to the root. B) Alignment quality criteria: These attributes are based on the BLAST alignments. For attributes B.1 - B.4, the best value for the corresponding attribute was taken, if a GO term occurred in more than one hit, B.1. Expectation value: the expectation value ("E-value") from BLASTX; B.2. Bit score: the bit score value provided by BLASTX; We wanted to award alignment length and quality by combining features. This was done with respect to the length of the query and the hits to offset biases due to different complexities of the query and subject organisms. Attributes B.3, B.4, C.3 and D.3 were obtained from initial trials with a small dataset (6270 cDNA sequences, data not shown) and applying parameter optimisation to distinguish the samples. B.3. Query coverage score (QCS): Combined measure of alignment size and quality concerning the query sequence, QCS := (AL / QL) × (I + S),     (5) where AL denotes the alignment length, QL the length of the query sequence, I the number of identities in the alignment, and S the number of positively contributing residues in the alignment; B.4. Subject coverage score (SCS): as in B.3, however only with respect to the corresponding subject sequence (database hit), SCS := (AL / SL) × (I + S),     (6) where SL denotes the length of the subject sequence; Additionally, we decomposed these attributes into the following further six attributes (B.5 - B.10). For these attributes, we considered the hit with the best coverage score if a GO term occurred in more than one hit (query coverage score for attributes B.5, B.7, B.9, and subject coverage score for B.6, B.8, B.10). B.5. Query percentage (QPC): Percentage of coverage of the alignment region in the query sequence (with respect to QCS), i.e. QPC := (AL / QL) × 100;     (7) B.6. Subject percentage (SPC) Percentage of coverage of the alignment region in the corresponding subject sequence (with respect to SCS), i.e. SPC := (AL / SL) × 100;     (8) B.7. Query identity (QI): Percentage of identical residues in the BLASTX alignment (with respect to QCS); B.8. Subject identity (SI): Percentage of identical residues in the BLASTX alignment (with respect to SCS); B.9. Query similarity (QS): Percentage of similar or positively contributing residues in the alignment (with respect to QCS); B.10.Subject similarity (SS): Percentage of similar or positively contributing residues in the alignment (with respect to SCS). C) GO frequency related attributes: We extracted information about the frequency of GO terms in the hits by the following attributes: C.1.GO frequency (FG): the number of hits that contained the sample GO term. C.2.Number of hits (TH): the total number of hits for the query. C.3. Frequency score (FS): the number of hits that contained the sample GO term. Unlike C.1, we limited this score to emphasize differences in queries with few hits: C.4.Species frequency: The number of organisms contributing to a sample GO term for a single query sequence; C.5.Total GO (TG): total number of GO terms from all hits. C.6. Unique GO (UG): as C.5, except, that GO terms occurring more than once (in the hits) were counted only once. D) GO frequency by considering relationships: For these attributes we applied the structure of the Gene Ontology graph. Not only perfectly matching terms were considered, but also their defined parents, children or siblings: D.1.Relative frequency for all (RA): the relationships for the sample GO term with all GO terms that occurred in the hits were calculated. The sum of non-"different" relationships i.e. parent, child, or sibling was used for this attribute; D.2.Relative frequency for unique (RU): similar to attribute D.1, with the exception that GO terms occurring more than once were counted only once. D.3.Relative frequency for all (limited) (RAlim): same as attribute D.1, however this score was limited to emphasize differences of queries with few hits: D.4.Relative frequency for unique (limited) (RUlim): same as attribute D.2, however this score was limited to emphasize differences of queries with few hits: E) Annotation quality related attributes: Quality attributes were selected from the evidence codes provided by the gene association tables of the GO-mapped sequence databases. We selected 9 commonly used evidence codes (TAS, NAS, ISS, IPI, IMP, IGI, IEP, IEA, IDA), resulting in attributes E.1 to E.9. The entries of these attributes for each sample GO term were calculated by summing the occurrences of the corresponding evidence codes of all hits. Training and testing the classifier Before training, normalisation was performed. We normalised the attributes by taking the logarithm (log) and log of log if necessary. We used log values for 16 attributes (B.3-B.10, C.3, C.4, D.1, D.2, D.4 and E.1) and log of log for 8 attributes (B.2, C.1, E.2, E.4-E.8). Furthermore, we converted the attribute values into mean 0 and standard deviation 1 by applying the Z-transformation. The feature table contained 856,632 samples and 31 attributes. We split the dataset into 99 training subsets. Each subset comprised of approximately 1% of the samples i.e. 8,566 GO terms. This resulted in 96 organism specific subsets and 3 hybrid subsets. We applied the support vector machines in the implementation of LIBSVM [36], which supports a weighted SVM for unbalanced data. We used a higher penalty (5 instead of 1) for false positives (FP) for the model selection and also the training process to support a high specificity of the resulting classifiers. Also note, that our training set contained a high portion of negative samples (see Table 1) due to our relaxed E-value threshold. We utilised the radial basis function kernel and set the parameter epsilon (tolerance of termination criterion) to 0.01. The parameter C (regularisation term, cost for false classification) and gamma (kernel width) of the SVM were optimised using a grid search. The grid search determined the combination of C (log2-range: 13 to 15, step 1) and gamma (log2-range: 10 to 15, step 1) with the lowest classification error according to a five-fold cross validation such that each of the 99 data subsets was subdivided into a training set (90%) and a validation set (10%). The validation sets were used to estimate the parameters C and gamma for each of the 99 classifiers individually. Finally, the parameters from the classifier selection were applied to train each of the classifiers with 90% of each data set, respectively. The testing was based on the same 13 organisms and 856,632 GO terms corresponding to 36,771 sequences as described above. We performed the testing by an organism-wise cross-validation so that one organism was used as test set and the remaining ones as the training set. Data availability The annotation for Xenopus laevis contig sequences is downloadable at . We followed the standard GO annotation style (using Gene ontology guideline). The evidence code is always IEA. The confidence value is included for each GO term. Authors' contributions The main work was carried out by AV. RK and KG conceived the idea of the study. AV and RK drafted the manuscript. FS developed and JM applied the machine learning strategy. KG implemented the databases in SRS. RE and SS supervised the work. All authors participated in reading, approving and revising the manuscript. Acknowledgments We thank the Gene Ontology Consortium and all groups that established GO association databases for making their data available through the web. This work was funded by the German Cancer Research Center (DKFZ), the Deutsche Krebshilfe and the Nationales Genom-Forschungs-Netz (NGFN). We also thank Suresh Kumar Swaminathan and Coral del Val for their suggestions during the preparation of the manuscript, and Andrea Mclntosh-suhr and Christopher Previti for proofreading. Figures and Tables Figure 1 A schematic representation of possible GO term relationships: A: GO1 is a "parent" of GO2 in a single path relationship. B: GO1 is a "parent" of GO2 in a multiple path relationship. C: GO1 is a "child" of GO2 in a single path relationship. D: GO1 is a "child" of GO2 in a multiple path relationship. E: GO1 and GO2 are "siblings" in a single path relationship. F: GO1 and GO2 are "siblings" in a multiple path relationship. MF denotes the molecular function node (root). Figure 2 General prediction scheme: The training sequences (S1) with known function (GOx, GOy, GOz) were searched across the protein databases, yielding hits with molecular function GO terms (GO1, GO2, GO3, GO4, GO5, GO6) and their features (see methods), sketched as dots in a two-dimensional feature space. If GO terms of the hits compared to GO terms of the query, they were classified as +1 (correct, green), and -1 otherwise (red). The classifier (SVM) separated the classes by an optimal separating hyperplane (OSH). Unknown sequences (S2) were searched in the same manner and the GO terms (GOn, GOm, GOo) were extracted. Their features were calculated and mapped into the feature space. The corresponding labels were assigned (correct/false). Figure 3 Accuracy and precision against the number of votes: The accuracy and precision values of the test data is plotted against the number of votes. An increasing number of votes increased the precision monotonically. Higher stringency yielded a sparse lowering of the accuracy due to the rate of false negatives. The relation between the precision and the number of votes was used for assigning confidence values for new predictions. Figure 4 ROC plots for the classifiers performance: ROC plots for the results of all organisms tested and the average of all test sequences. The classification performance for different classes of organisms like multi-cellular eukaryotes, single-cell eukaroyotes and the prokaryotes were compared. Figure 5 Precision against the sequence coverage: Average precision against sequence coverage for all 13-test organisms (circles). The red line denotes a fitting curve. Figure 6 Comparison of GO slims between Xenopus, fly, yeast and mouse: Distributions of higher-level GO terms (,,GO slim", see text) for Xenopus, fly, yeast and mouse. The sum of all high-level terms may exceed the total number of the annotated terms, since some terms may have more than one high-level "parent" terms due to multiple paths. Table 1 Training and test dataset: Datasets used for training and testing the support vector machines. The columns are: 1. The number of cDNA sequences for training. 2. The number of cDNA sequences with BLAST hits having GO molecular function terms. 3. The average number of GO molecular function terms per cDNA sequence of the BLAST-hits. 4. The classification of GO terms coming from the hits, positive if the GO terms were similar to original annotation, negative otherwise. Organisms Number of cDNAs cDNA with MF GO Number of GO/cDNA Class distribution % Positive % Negative Rat 1039 1036 36.90 25.7 74.3 Fish 1061 1044 32.10 39.2 60.8 Fly 5840 5574 25.47 23.4 76.6 Worm 4272 3458 27.13 39.5 60.5 Plasmodium 274 271 23.67 28.0 72.0 Leishmania 82 82 20.51 35.1 64.9 Yeast 3356 2972 18.60 23.7 76.3 Bacillus 2729 2577 13.63 35.4 64.6 Coxiella 931 900 12.33 37.0 63.0 Shewanella 2413 2303 10.78 33.0 67.0 Vibrio 1832 1804 12.54 31.9 68.1 Arabidopsis 8807 8120 26.66 30.2 69.8 Table 2 The novel predictions with high confidence values (100%). The columns correspond to TC (TIGR-id) number, GO term level in GO-structure, the confidence values and the GO number and GO terms. TC Numbers GO level Confidence value GO ID GO terms TC212171 4 100% GO:0008233 peptidase activity 5 100% GO:0004175 endopeptidase activity 5 98% GO:0008236 serine-type peptidase activity 3 98% GO:0016787 hydrolase activity TC196381 5 100% GO:0004175 endopeptidase activity 4 98% GO:0008233 peptidase activity 3 98% GO:0016787 hydrolase activity 5 97% GO:0008236 serine-type peptidase activity TC209487 2 100% GO:0003824 enzyme activity 3 100% GO:0016787 hydrolase activity 5 90% GO:0004177 aminopeptidase activity 4 85% GO:0017171 serine hydrolase activity TC187949 4 100% GO:0004888 transmembrane receptor activity 3 97% GO:0004872 receptor activity TC194305 2 100% GO:0003824 enzyme activity 3 99% GO:0016740 transferase activity 3 99% GO:0016301 kinase activity 6 97% GO:0004672 protein kinase activity TC210151 3 100% GO:0004872 receptor activity 4 97% GO:0004888 transmembrane receptor activity 6 82% GO:0004928 frizzled receptor activity TC199713 6 100% GO:0004602 glutathione peroxidase activity 3 98% GO:0016491 oxidoreductase activity 5 85% GO:0004601 peroxidase activity TC190605 2 100% GO:0003824 enzyme activity 3 100% GO:0016787 hydrolase activity 4 87% GO:0017171 serine hydrolase activity 6 86% GO:0016804 prolyl aminopeptidase activity ==== Refs Lewis S Ashburner M Reese MG Annotating eukaryote genomes Curr Opin Struct Biol 2000 10 349 354 10851187 10.1016/S0959-440X(00)00095-6 Searls DB Using bioinformatics in gene and drug discovery Drug Discov Today 2000 5 135 143 10729819 10.1016/S1359-6446(99)01457-9 Bork P Koonin EV Predicting function from protein sequence: Where are the bottlenecks? Nat Genet 1998 18 313 318 9537411 10.1038/ng0498-313 Smith TF Functional genomics – bioinformatics is ready for the challenge Trends Genet 1998 14 291 293 9676532 10.1016/S0168-9525(98)01508-X Bork P Gibson TJ Applying motif and profile searches Methods Enzymol 1996 266 162 184 8743684 10.1016/S0076-6879(96)66013-3 Andrade MA Brown NP Leroy C Hoersch S de Daruvar A Reich C Franchini A Tamames J Valencia A Ouzounis C Sander C Automated genome sequence analysis and annotation Bioinformatics 1999 15 391 412 10366660 10.1093/bioinformatics/15.5.391 Bork P Bairoch A Go hunting in sequence databases but watch out for the traps Trends Genet 1996 12 425 427 8909140 10.1016/0168-9525(96)60040-7 Galperin MY Koonin EV Sources of systematic errors in functional annotation of genomes: domain rearrangements, non-orthologous gene displacement, and operon distribution In Silico Biol 1998 1 0007 Sakata K Nagamura Y Numa H Antonio BA Nagasaki H Idonuma A Watanabe W Shimizu Y Horiuchi I Matsumoto T Sasaki T Higo K RiceGAAS: an automated annotation system and database for rice genome sequence Nucleic Acids Res 2002 30 98 102 11752265 10.1093/nar/30.1.98 Bailey LC Fischer S JrSchug J Crabtree J Gibson M Overton GC GAIA: framework annotation of genomic sequence Genome Res 1998 8 234 250 9521927 Harris NL Genotator: a workbench for sequence annotation Genome Res 1997 7 754 762 9253604 Gaasterland T Sensen CW MAGPIE: automated genome interpretation Trends Genet 1996 12 76 78 8851977 10.1016/0168-9525(96)81406-5 Kitson DH Badretdinov A Zhu ZY Velikanov M Edwards DJ Olszewski K Szalma S Yan L Functional annotation of proteomic sequences based on consensus of sequence and structural analysis Brief Bioinform 2002 3 32 44 12002222 10.1186/1471-2105-3-32 Frishman D Albermann K Hani J Heumann K Metanomski A Zollner A Mewes HW Functional and structural genomics using PEDANT Bioinformatics 2001 17 44 57 11222261 10.1093/bioinformatics/17.1.44 The Gene Ontology Consortium Gene Ontology: tool for the unification of biology Nat Genet 2000 25 25 29 10802651 10.1038/75556 The Gene Ontology Consortium Creating the gene ontology resource: design and implementation Genome Res 2001 11 1425 1433 11483584 10.1101/gr.180801 Hill DP Blake JA Richardson JE Ringwald M Extension and Integration of the Gene Ontology (GO): Combining GO vocabularies with external vocabularies Genome Res 2002 12 1982 1991 12466303 10.1101/gr.580102 Xie H Wasserman A Levine Z Novik A Grebinskiy V Shoshan A Mintz L Large-Scale Protein Annotation through Gene Ontology Genome Res 2002 12 785 794 11997345 10.1101/gr.86902 Camon E Magrane M Barrell D Binns D Fleischmann W Kersey P Mulder N Oinn T Maslen J Cox A Apweiler R The Gene Ontology Annotation (GOA) project: implementation of GO in SWISS-PROT, TrEMBL, and InterPro Genome Res 2003 13 662 672 12654719 10.1101/gr.461403 TIGR Gene Indices Zehetner G OntoBLAST function: from sequence similarities directly to potential functional annotations by ontology terms Nucleic Acids Res 2003 31 3799 3803 12824422 10.1093/nar/gkg555 Hennig S Groth D Lehrach H Automated Gene Ontology annotation for anonymous sequence data Nucleic Acids Res 2003 31 3712 3715 12824400 10.1093/nar/gkg582 Jensen LJ Gupta R Staerfeldt HH Brunak S Prediction of human protein function according to Gene Ontology categories Bioinformatics 2003 19 635 642 12651722 10.1093/bioinformatics/btg036 Schug J Diskin S Mazzarelli J Brunk BP Stoeckert CJ Jr Predicting Gene Ontology Functions from ProDom and CDD Protein Domains Genome Res 2002 12 648 655 11932249 10.1101/gr.222902 Bauer E Kohavi R An Empirical Comparison of Voting Classification Algorithms: Bagging, Boosting, and Variants Machine Learning 1999 36 105 139 10.1023/A:1007515423169 Peiffer DA Cho KWY Shin Y Xenopus DNA Microarrays Current Genomics 2003 4 665 672 Altschul SF Madden TL Schäffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389 TIGR Xenopus laevis Gene Index Release 7.0; January 8, 2004 Gene Ontology Consortium Hand D Mannila H Smyth P Principles of Data Mining, 2001 MIT Press, Cambridge, London Hastie T Tibshirani R Friedman J The Elements of Statistical Learning, 2001 Springer, New York, Berlin, Heidelberg Swiss-Prot Senger M Flores T Glatting K Ernst P Hotz-Wagenblatt A Suhai S W2H: WWW interface to the GCG sequence analysis package Bioinformatics 1998 14 452 457 9682058 10.1093/bioinformatics/14.5.452 Ernst P Glatting KH Suhai S A task framework for the web interface W2H Bioinformatics 2003 19 278 282 12538250 10.1093/bioinformatics/19.2.278 Del Val C Glatting KH Suhai S cDNA2Genome: A tool for mapping and annotating cDNAs BMC Bioinformatics 2003 4 39 12964951 10.1186/1471-2105-4-39 LIBSVM; version 2.4

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==== Front Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-81535788210.1186/1465-9921-5-8ResearchIn vivo clearance of surfactant lipids during acute pulmonary inflammation. Malloy Jaret L [email protected] Jo Rae [email protected] Address: Box 3709, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA2004 23 7 2004 5 1 8 8 30 1 2004 23 7 2004 Copyright © 2004 Malloy and Wright; licensee BioMed Central Ltd.2004Malloy and Wright; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background A decrease in pulmonary surfactant has been suggested to contribute to the lung dysfunction associated with pulmonary inflammation. A number of studies have implicated surfactant clearance as a possible mechanism for altered pool sizes. The objective of the current study was to specifically investigate the mechanisms of surfactant clearance in a rodent model of acute pulmonary inflammation. Methods Inflammation was induced by intrapulmonary instillation of lipopolysaccharide (LPS: 100 μg/kg). Lipid clearance was assessed at 18 and 72 hours post-LPS instillation by intratracheal administration of radiolabel surfactant-like liposomes 2 hours prior to isolation and analysis of inflammatory cells and type II cells. Results At both 18 and 72 hours after LPS instillation there was significantly less radioactivity recovered in the lavage fluid compared to respective control groups (p < 0.05). At both time points, the number of cells recovered by lavage and their associated radioactivity was greater compared to control groups (p < 0.01). There was no difference in recovery of radioactivity by isolated type II cells or other cells obtained from enzymatic digestion of lung tissue. Conclusion These results show that increased clearance of surfactant lipids in our model of acute pulmonary inflammation is primarily due to the inflammatory cells recruited to the airspace and not increased uptake by alveolar type II cells. ==== Body Background Pulmonary surfactant is a phospholipid-protein complex that lines the inner surface of the lung and is essential for normal pulmonary function. Surfactant acts to promote lung stability by reducing surface tension within the lung, while also protecting against inhaled pathogens. Surfactant is composed of approximately 90% lipids and 10% proteins by weight. The lipid component is primarily phospholipids with phosphatidylcholine (PC) being the most abundant, and the protein component comprised of four surfactant-associated proteins designated SP-A, SP-B, SP-C and SP-D [1]. The reduction of surface tension within the lung is a result of the interaction between surfactant phospholipids and the two hydrophobic surfactant proteins, SP-B and SP-C [1], while the two hydrophilic proteins, SP-A and SP-D, are members of a family of innate immune molecules called collectins [2]. Collectins opsonize bacteria and viruses and enhance their phagocytosis by macrophages and neutrophils [2]. Alterations of the pulmonary surfactant system, including decreased total surfactant levels, have been implicated in the pathophysiology of acute lung injury. Multiple studies of patients with a variety of lung diseases have shown that surfactant levels are decreased in the inflamed, injured, or infected lung [3-5]. In agreement, decreases in alveolar surfactant lipid pools have also been observed in several animal models of lung inflammation induced by both direct insults to the lung, such as bacterial infection [6], oxygen toxicity [7,8], endotoxin administration [9-11], and by indirect insults, such as N-nitroso-N-methylurethane [12] and cecal ligation and perforation [13]. Alveolar metabolism of surfactant is a complex process, primarily involving type II epithelial cells that synthesize, secrete and clear surfactant from the airspaces [14], along with phagocytic cells such as macrophages and neutrophils that participate in surfactant clearance [15,16]. In a situation of pulmonary inflammation, altered type II cell metabolism has been thought to play a role in the alterations of surfactant lipid levels. Viviano et al. observed a decrease in alveolar surfactant levels and a corresponding increase in intracellular surfactant after lipopolysaccharide (LPS) administration [10]. Additionally, exposure of rat lungs ventilated ex vivo to LPS resulted in the presence of giant lamellar bodies within the type II cells [17,18]. Collectively these studies suggest that type II cell metabolism is altered after LPS administration and that a possible explanation for decreased surfactant pool size may be increased clearance of surfactant lipids by the type II cells. Additional experimental evidence has also implicated recruited inflammatory cells as having an impact on surfactant pool sizes. Both neutrophils and macrophages recovered from LPS exposed lungs had a greater capacity to internalize surfactant-like lipids compared to control cells in vitro [16]. Therefore these recruited inflammatory cells may also have a significant impact on surfactant pool size by increasing the overall surfactant lipid clearance. The objective of the current study was specifically to investigate the mechanisms of in vivo surfactant clearance in a rodent model of acute pulmonary inflammation induced by intrapulmonary instillation of LPS. We hypothesized that during pulmonary inflammation there is altered clearance of alveolar phospholipid by both alveolar type II cells and inflammatory cells within the airspaces. We analyzed in vivo clearance of surfactant lipids by a variety of pulmonary cells at 18 and 72 hours after LPS instillation. Results indicated that increased clearance of surfactant lipids in our model of acute pulmonary inflammation is primarily due to enhanced lipid uptake by the inflammatory cells recruited to the airspace, whereas uptake by type II cells was unaltered. However the in vivo surfactant uptake by inflammatory cells in the current study was significantly less than that predicted by previous in vitro studies [16]. Methods Materials Dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), dipalmitoylphosphatidylglycerol (DPPG) and cholesterol were purchased from Avanti Polar Lipids (Birmingham, AL). L-α-dipalmitoyl [2-palmitoyl-9,10-3H(N)]PC was obtained from DuPont New England Nuclear (Boston, MA). Elastase for type II cell isolations was purchased from Worthington Biochemicals (Freehold, NJ). Dulbecco's PBS, DMEM, and fetal bovine serum (FBS) were obtained from Life Technologies (Gaithersburg, MD). Low-endotoxin BSA, O26:B6 Escherichia coli LPS, Rat IgG and all other chemicals were purchased from Sigma (St. Louis, MO). Chloroform and methanol were obtained from EM Science (Gibbstown, NJ). Preparation of Liposomes Small unilamellar liposomes were prepared with a lipid composition similar to pulmonary surfactant: 52% DPPC, 26% egg PC, 15% DPPG and 7% cholesterol by weight with trace amounts of 3H-DPPC (12 μCi/mg phospholipid). The lipids were dried under nitrogen, reconstituted in 0.15 M saline and extruded from a French Press cell under 900 psi. This resulted in small unilamellar liposomes at a concentration of approximately 1 mg lipid/ml. Animal Model Male pathogen-free Sprague Dawley rats (150–200 g; Taconic Farms, Germantown, NY) were used for the current study. For LPS animals, endotoxin (0.1 mg/kg of O26:B6 E. coli LPS) was suspended in 300 μl of sterile 0.15 M saline. Control animals received an equal volume (300 μl) of sterile 0.15 M saline. For instillation, animals were anesthetized with halothane such that they remained unconscious throughout the entire instillation procedure and had no cough reflex upon intubation. Animals were placed on a board at a 45° angle, intubated with an 18-gauge blunt ended catheter and either sterile saline or LPS suspension was instilled followed by five 1 ml boluses of air to facilitate the distribution of the instilled fluid. Two hours prior to killing, liposomes (100 μg lipid) were intratracheally instilled in all groups following the identical instillation procedure. Separate groups of both control and LPS animals were sacrificed at 18 or 72 hours after saline or LPS instillation. Whole Lung Studies One control animal and one LPS animal were investigated simultaneously, thus all procedures were done in parallel. At 18 or 72 hours after saline or LPS instillation, animals were anesthetized with 0.3 mg of sodium pentobarbital and 700 U of heparin. After loss of toe pinch reflex, the trachea was cannulated and the rat was exsanguinated via transection of the descending aorta. The chest cavity was subsequently opened and the lungs perfused through the pulmonary artery with 40–50 ml of a calcium buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 10 mM HEPES, 2.0 mM CaCl2, and 1.3 mM MgSO4 at 37°C). The lung from the first animal was carefully removed from the thoracic cavity and placed between saline soaked gauze pads while the second lung was perfused and removed in identical fashion. The time for lung perfusion and removal was approximately 10 minutes. After lungs were isolated from both a control and LPS animal, they were lavaged simultaneously with eight 10-ml volumes of EGTA buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na2HPO4, 10 mM HEPES, and 0.2 mM EGTA at 37°C). For each animal the individual lavages were collected and combined to make up the bronchoalveolar lavage fluid (BALF), which was immediately stored on ice. After the lavage procedure, individual lobes were dissected away from the major airways. The lung tissue was then cut into 5 mm pieces in 5 ml calcium buffer and subsequently homogenized with a Polytron PT-MR-2100. After complete homogenization, total lung tissue volume was diluted to 40 ml with calcium buffer and stored on ice. A small aliquot (2 ml) of the total BALF was removed and the remainder was centrifuged at 250 g for 10 minutes at 4°C to generate a pellet that was primarily BALF cells. The cell pellet was subsequently suspended in 10 ml of PBS. Lavage cell numbers were determined by a hemocytometer, viability determined by trypan blue exclusion and cell differential determined by Hemacolor staining of cytospins that were prepared using a Shandon Cytospin 2 centrifuge. Liposome-association in total BALF, cell-free BALF, BALF cells and homogenized lung tissue was determined by scintillation counting. Surfactant Phospholipid Measurement Alveolar phospholipid levels were measured in the cell-free BALF by phospholipid-phosphorous measurement. Lipids were extracted using the method of Bligh & Dyer [19] and phospholipid levels were determined using a modification of the Duck-Chong phosphorous assay [20]. Briefly, 100 μl of 10% magnesium nitrate in methanol was added to the extracted lipids. After drying, the samples were ashed in a fume hood on an electric rack for approximately 1 min. After 1 ml of 1 M HCl was added, the samples were reheated on a heating block while covered for 15 min at 95°C. After cooling, a 66 μl aliquot of each sample was added to individual wells of a 96 well plate along with 134 μl of a dye consisting of 4.2% ammonium molybdate in 4.5 M HCl with 0.3% malachite green (1:3 vol/vol). The absorbency of each sample was read at 650 nm using a Biorad 550 microplate reader and compared to reference standards on the same plate. Surfactant Protein A (SP-A) Analysis Relative quantities of alveolar SP-A from 18-hour control animals, 18-hour LPS animals and 72-hour LPS animals were determined by loading equal volumes of BALF on 15% SDS-PAGE gels under reducing conditions. Total BALF recovery was not different among the individual animals. Proteins were then transferred to nitrocellulose where SP-A was probed with a well characterized polyclonal rabbit anti-rat SP-A [21]. The nitrocellulose was subsequently developed using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). Type II Cell Isolation A separate cohort of animals was required for type II cell isolation studies. As with the whole lung studies, type II cell isolation was completed for one control and one LPS rat in parallel for both 18- and 72-hour time points after instillation of saline or LPS. The type II cell isolation procedure was described previously with minor modifications of the original protocol [22,23]. The modifications were designed for isolation of type II cells from inflamed lungs, and included increasing the elastase to 3,000 orcein units/lung, and increasing the surface area for IgG panning by two-fold. Briefly the isolation procedure involved killing the rats, removing the lungs and lavaging the lungs eight times as stated in Whole Lung Studies. Subsequently the lungs were lavaged twice with calcium buffer (37°C) and once with elastase solution (3000 orcein/40 ml calcium buffer; 37°C). The lungs were then filled with the elastase solution and suspended in warmed saline (37°C) for 20 minutes. Lung tissue was removed from the major airways, cut into 5-mm pieces and chopped 200 times with sharp scissors in 5 ml of the calcium solution along with 2 mg of DNase. The tissue suspension was added to a flask with 4 ml of FBS and 35 ml of calcium buffer and shaken vigorously for 2 min in a 37°C water bath. The resultant tissue suspension was strained through gauze, and then decreasing sizes of nylon mesh (150-, 15-, and 8-μm). The cell suspension was centrifuged at 250 g for 10 min at 4°C, the resulting cellular pellet was suspended in 20 ml of warm DMEM (37°C) and incubated on two IgG coated Petri dishes (150 × 15 mm) for 30 minutes at 37°C. After the incubation period, nonadherent cells were removed from the plate, washed once with PBS, suspended in calcium buffer and used for determination of radiolabel recovery associated with Type II cells. The remaining adherent cells were gently scraped off the plate in 5 ml of DMEM and transferred into a polypropylene tube; this population of cells is referred to as "plate cells" and consisted primarily of macrophages and/or neutrophils as described in detail below. The cells were washed once and suspended in calcium buffer for determination of associated radioactivity. A small aliquot from the isolated type II cells and plate cells was saved for differential cell count and viability. Cell purity was determined by counting a minimum of 250 cells from random fields after staining by the Papanicolaou method [22]. Statistics All data reported are means ± standard error (SE). Repetitions (n) used to calculate means ± SE were from independent experiments, not from replicates within an experiment. An analysis of variance (ANOVA) was used to determine differences between all experimental groups at a specific time point, followed by a Tukey post-hoc test for multiple comparisons. Significance was accepted when p < 0.05. Results LPS-induced alterations: BALF cells and alveolar surfactant Table 1 reveals total alveolar cell numbers and differentials from the BALF of control and LPS groups killed at the 18- and 72-hour time points. Eighteen hours after instillation of LPS there were significantly more cells recovered in the BALF from the LPS treated animals compared to the saline control group (p < 0.01). Cell differentials from the two 18-hour groups revealed that the LPS group had primarily neutrophils in the BALF, whereas the cells recovered from the control group were predominantly macrophages. Seventy-two hours after LPS instillation there were significantly greater numbers of cells in the BALF of the LPS group compared to the 72-hour control group (p < 0.01), but significantly fewer numbers of cells compared to the LPS 18-hour group (p < 0.01). Cell differentials from the 72-hour groups revealed that the cells were predominately macrophages in both the LPS and control groups. Of note, there were no differences in cell numbers or differentials between the two control groups at the different time points. Table 1 Bronchoalveolar lavage fluid characteristics from animals killed 18 and 72 hours after instillation of saline (Control) or lipopolysaccharide (LPS). Control 18 (n = 6) LPS 18 (n = 6) Control 72 (n = 6) LPS 72 (n = 6) Cell Numbers (106) 12.3 ± 1.1 102.8 ± 8.1* 11.3 ± 0.9 57.3 ± 6.6*# % Macrophages 98.2 ± 0.3 7.3 ± 0.8* 97.7 ± 0.4 90 ± 1.5* % Neutrophils 1.8 ± 0.3 92.7 ± 0.8* 2.3 ± 0.4 10 ± 1.5* Alveolar Phospholipid (mg PL/kg BW) 14.7 ± 0.7 12.8 ± 0.9 14.0 ± 0.4 12.9 ± 0.5 Abbreviations: PL = phospholipid; BW = body weight. Values are mean ± SEM. * = p < 0.01 vs. appropriate control; # = p < 0.01 vs. LPS 18. Table 1 also displays the total phospholipid levels measured in the BALF of the four experimental groups. There was an approximate 15% decrease in total phospholipid recovered by lung lavage from both LPS groups compared to their respective saline control groups, however this did not reach statistical significance. Of note, there was no difference in mean body weights among the four experimental groups (Con 18: 177 ± 7 g; LPS 18: 180 ± 10 g; Con 72: 186 ± 9 g; LPS 72: 192 ± 7 g). Figure 1 shows Western blot analysis of SP-A measured in the BALF recovered from animals killed 18 hours post saline instillation (control), and 18 hours and 72 hours after LPS instillation. There was relatively greater quantity of SP-A in the BALF of animals killed at both 18 and 72 hours after LPS instillation compared to the control animals, consistent with previous reports using similar models of intrapulmonary LPS administration [9-11]. Figure 1 Alveolar surfactant protein A (SP-A) levels. Relative quantities of alveolar SP-A were measured by Western Blot. Equal volumes of bronchoalveolar lavage fluid (BALF) from the individual animals were utilized and total BALF recovery was not different among these animals. Lanes 1 and 2 represent individual animals that were lavaged 18 hours after saline instillation. Lanes 3 and 4 represent individual animals that were lavaged 18 hours after instillation of 100 μg/kg O26:B6 lipopolysaccharide (LPS). Lanes 5 and 6 represent animals lavaged 72 hours after instillation of 100 μg/kg 026:B6 LPS. Whole Lung Studies: Clearance of Radiolabel Liposomes Figure 2 shows distribution of the recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 18 hours after instillation of saline or LPS. There was no difference in total lung radioactivity recovered compared to the total radiolabel instilled between the Control 18 and LPS 18 groups (46.0 ± 4.6% and 48.4 ± 4.0%; respectively). There was significantly less radiolabel recovered in the BALF (p < 0.05) and significantly more radiolabel associated with the BALF cells (p < 0.01) in the LPS group compared to the control group. There was no difference in radiolabel association with lung tissue between the two groups. Figure 2 Distribution of radiolabel liposomes 18 hours after instillation of LPS. Whole lung distribution of the total recovered 3H-liposomes instilled 2 hours prior to killing. Animals were killed 18 hours after instillation of saline (Con 18) or 100 μg/kg O26:B6 lipopolysaccharide (LPS 18) where the lungs were lavaged, lavage cells isolated and whole lung tissue homogenized. Radioactivity was subsequently measured in cell free bronchoalveolar lavage fluid (BALF), isolated BALF cells and whole lung tissue homogenate (Tissue). Data are means ± SEM and expressed as a percentage of total recovered radiolabel; n = 6 animals/group. Statistical significance * = p < 0.05 vs. respective control group. Figure 3 displays total recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 72 hours after instillation of saline or LPS. There was no difference in the total lung radiolabel recovered compared to the total radiolabel instilled between the Control 72 and LPS 72 groups (47.3 ± 4.0% and 44.8 ± 3.8%; respectively). There was significantly less radiolabel recovered in the BALF (p < 0.05) and significantly more radiolabel liposomes associated with the BALF cells (p < 0.01) in the LPS group compared to the control group. There was no difference in radiolabel associated with lung tissue between the two groups. Figure 3 Distribution of radiolabel liposomes 72 hours after instillation of LPS. Whole lung distribution of the total recovered 3H-liposomes instilled 2 hours prior to killing. Animals were killed 72 hours after instillation of saline (Con 72) or 100 μg/kg O26:B6 lipopolysaccharide (LPS 72) where the lungs were lavaged, lavage cells isolated and whole lung tissue homogenized. Radioactivity was subsequently measured in cell free bronchoalveolar lavage fluid (BALF), isolated BALF cells and whole lung tissue homogenate (Tissue). Data are means ± SEM and expressed as a percentage of total recovered radiolabel; n = 6 animals/group. Statistical significance * = p < 0.05 vs. respective control group. Additional controls were performed in which animals (n = 2) received radiolabel liposomes followed by whole lung lavage and lung tissue homogenization 5 minutes after the instillation procedure. Mean total lung radiolabel recovery for this control group was 77% suggesting that ~25% of the radiolabel liposomes were lost in the instillation procedure (i.e., syringe, catheter), or possibly were adhered to the airway epithelium. The distribution of the recovered liposomal radioactivity within the 5-minute control lungs was 91% associated with the BALF, 2% associated with BALF cells, and 7% associated with lung tissue. Type II Cell Isolation: Cell Recoveries, Purities and Radioactivity per Cell Table 2 demonstrates the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 18-hour time point. Type II cell recovery for the control group ranged from 10.4 × 106 to 21.6 × 106 cells with a mean of 17.1 × 106. The purity of this cell fraction averaged greater than 80% type II cells and viability was greater than 95%. Plate cell recovery from the control group ranged from 1.8 × 106 to 4.7 × 106 cells with a mean value of 3.2 × 106. This cell fraction was predominately macrophages and the viability was greater than 90%. For the LPS group at the 18-hour time point, type II cell recovery ranged from 10.6 × 106 to 20.5 × 106 cells and a mean of 16.3 × 106 cells, with a purity greater than 85% type II cells and viability greater than 95%. Plate cells from the LPS 18 group ranged from 2.7 × 106 to 13 × 106 cells with a mean of 6.6 × 106 cells. This cell population consisted primarily of neutrophils and macrophages and viability was greater than 90%. There were no significant differences in cell numbers obtained from control and LPS animals 18 hours after saline or LPS instillation. Table 2 Cell recovery and differential after lung digestion and IgG panning for animals killed 18 hours after instillation of saline (Control) or lipopolysaccharide (LPS). Control 18 (n = 7) LPS 18 (n = 7) Type II Cells Plate Cells Type II Cells Plate Cells Cell Numbers (106) 17.1 ± 1.6 3.2 ± 0.5 16.3 ± 1.8 6.6 ± 1.7 % Type II 81 ± 2 15 ± 7 86 ± 2 14 ± 2 % Macrophages 9 ± 1 61 ± 5 3 ± 1* 33 ± 2* % Neutrophils 1 ± 1 17 ± 10 4 ± 1 53 ± 3* % Other Cells 9 ± 2 7 ± 2 7 ± 2 - Values are mean ± SEM. * = p < 0.01 vs. Control. Figure 4 reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue associated cells (plate cells) for the control and LPS 18-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the control group and the LPS group at the 18-hour time point after instillation. Figure 4 Liposome uptake by lung tissue cells 18 hours after instillation of LPS. Association of 3H-liposomes instilled 2 hours prior to killing in cells isolated from lung tissue. Animals were killed 18 hours after instillation of saline (Con 18) or 100 μg/kg O26:B6 LPS (LPS 18). Lungs were lavaged and type II cells and other tissue-associated cells (plate cells) were isolated. Data are means ± SEM and expressed as DPM/106 cells, n = 7 animals/group. Table 3 shows the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 72-hour time point. Type II cell recovery for the control group ranged from 21.2 × 106 to 40 × 106 cells with a mean of 28.4 × 106. The purity of this cell fraction averaged 75% type II cells and viability was greater than 95%. The number of cells obtained from the plate group ranged from 1.8 × 106to 15.9 × 106 cells with a mean value of 6.2 × 106 with the primary cell type being macrophages with a small percentage of neutrophils and type II cells. Viability for the plate cells were greater than 90%. For the LPS group at the 72-hour time point, type II cell recovery ranged from 19.2 × 106 to 64.6 × 106 cells and a mean of 33.2 × 106 cells, purity greater than 80% type II cells and viability greater than 95%. Plate cells from the LPS 18-hour group ranged from 3.3 × 106 to 16.2 × 106 cells with a mean of 7.8 × 106 cells. This cell population consisted primarily of macrophages and viability was greater than 90%. There were no significant differences in cell recoveries between the control and LPS groups at the 72-hour time point. Of note, there were a greater number of cells recovered from both 72-hour groups compared to both 18-hour groups. Table 3 Cell recovery and differential after lung digestion and IgG panning for animals killed 72 hours after instillation of saline (Control) or lipopolysaccharide (LPS). Control 72 (n = 6) LPS 72 (n = 7) Type II Cells Plate Cells Type II Cells Plate Cells Cell Numbers (106) 28.4 ± 3.9 6.2 ± 2.2 33.2 ± 5.6 7.8 ± 2.2 % Type II Cells 75 ± 2 22 ± 3 83 ± 1 19 ± 3 % Macrophages 11 ± 2 56 ± 5 12 ± 2 59 ± 4 % Neutrophils - 22 ± 7 2 ± 1 22 ± 3 % Other Cells 14 ± 1 - 3 ± 3* - Values are mean ± SEM. * = p < 0.01 vs. Control. Figure 5 reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue-associated cells (plate cells) for the control and LPS 72-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the LPS group and the control group 72 hours after the appropriate instillation. Of note there was no significant difference in radioactivity per cell for both the type II cells and plate cells between the two 72-hour groups and the two 18-hour groups. Figure 5 Liposome uptake by lung tissue cells 72 hours after instillation of LPS. Association of 3H-liposomes instilled 2 hours prior to killing in cells isolated from lung tissue. Animals were killed 72 hours after instillation of saline (Con 72) or 100 μg/kg O26:B6 LPS (LPS 72). Lungs were lavaged and type II cells and other tissue-associated cells (plate cells) were isolated. Data are means ± SEM and expressed as DPM/106 cells; Con 72 = 6 animals, LPS 72 = 7 animals. Discussion In the present study, we evaluated the clearance of surfactant-like liposomes at 18 and 72 hours following the intrapulmonary instillation of LPS. At both time points, radiolabel liposomes were instilled two hours prior to killing and the distribution within the lung was determined. Radioactivity was measured in cell-free BALF, isolated BALF cells, whole lung tissue, isolated type II cells and remaining tissue-associated cells. At both time points there was a significant increase in clearance of exogenous liposomes from the airspace and a small decrease in alveolar surfactant phospholipid levels in the LPS groups compared to control groups that received vehicle only. This corresponded with increased radiolabel associated with isolated BALF cells and no difference associated with lung tissue at either time point after LPS instillation compared to controls. There was no difference in liposomal radioactivity associated with isolated type II cells or other tissue cells between both LPS groups and their respective control groups, which was in agreement with whole lung measurements. These data suggest that cells recruited into the airspace as a consequence of the LPS-induced inflammatory response can significantly contribute to increased clearance of surfactant-like liposomes from the airspace (Figs. 2 and 3). Eighteen hours after LPS instillation there was an infiltration of neutrophils into the airspace which resulted in a 10-fold increase in BALF cells. At this time point there was a doubling of total liposomal radioactivity associated with these inflammatory cells compared to alveolar cells recovered from control animals. Neutrophils recruited into alveolar spaces during infection are essential for host defense by phagocytosis and killing of bacteria and other infectious agents. Previous studies have documented that surfactant can in fact modulate neutrophil functions, as surfactant proteins A and D can enhance neutrophil uptake of bacteria [24]. Although these phagocytic cells are extremely important in host defense, they have also been implicated as a contributing factor to the lung injury in a variety of inflammatory disorders. In a recent study by Quintero et al., it was demonstrated by confocal microscopy that neutrophils isolated from inflamed lungs were able to significantly internalize and degrade surfactant lipids in vitro [16]. Based on in vitro measurements of lipid uptake, they estimated that neutrophils could account for up to 48% of the observed clearance and could significantly impact surfactant homeostasis. Data from the current study supports this idea by demonstrating that in vivo, neutrophils can indeed contribute to the clearance of surfactant lipids during pulmonary inflammation. At the 72-hour time point after LPS instillation there was an accumulation of macrophages in the alveolar space that resulted in a 5-fold increase in BALF cells compared to control animals. There was a doubling of radiolabeled phospholipids associated with these alveolar cells and no increase in the amount of radiolabeled phospholipids associated with whole lung tissue. This resulted in a significant increased in clearance of total liposomal radioactivity from the BALF, 72 hours after LPS instillation. Alveolar macrophages have been shown in vitro to take up and degrade surfactant lipids and have also been shown in vivo to account for approximately 20% of surfactant lipid clearance in normal lungs [16,25]. Macrophages isolated from LPS-exposed lungs were shown to have a greater capacity to take up surfactant lipids in vitro than macrophages isolated from normal lungs [16], suggesting that in an inflamed lung, the recruited macrophages may have a significant role in surfactant metabolism. Indeed, the in vivo data presented in the current study support the theory that activated alveolar macrophages can impact clearance of surfactant lipids. Of note, previous in vitro data had predicted that inflammatory cells (neutrophils and macrophages) could account for a 6 to 13 fold increase in lipid clearance [16]. In the current study there was a 5–10 fold increase in alveolar inflammatory cells but only a doubling of cell associated radioactivity and a change in alveolar phospholipid pool size of approximately 15%. Importantly, the current study instilled a similar dose of LPS, had similar end points and the same number of inflammatory cells recovered from the BALF as the aforementioned in vitro study. Although the present in vivo data demonstrated that the inflammatory cells can indeed contribute to increased lipid clearance as suggested previously, the absolute value was considerably less than that predicted from the in vitro studies. Although we do not have an explanation for this quantitative difference between the in vivo and in vitro observations, it is possible that the process of cell isolation in the in vitro study resulted in their activation for subsequent lipid uptake. Alternatively, in vivo, multiple factors can impact total surfactant pool size in addition to clearance by inflammatory cells. Type II cells are the predominant cell type that regulates surfactant metabolism, being involved in synthesis, secretion and clearance. Secretion of surfactant phospholipids is solely a property of alveolar type II cells, whereas clearance in a healthy lung is regulated primarily by both type II cells and alveolar macrophages. In one study utilizing uninjured rabbits, it was determined that type II cells accounted for approximately 65% of the clearance of alveolar phospholipid [25]. In a situation of pulmonary inflammation induced by LPS administration, there have been documented decreases in alveolar surfactant phospholipids [9-11], increases in intracellular surfactant phospholipids [10] and the appearance of giant lamellar bodies within type II cells [17,18,26]. From these observations, LPS induced inflammation could either inhibit surfactant secretion or enhance surfactant clearance by type II cells, resulting in diminished alveolar surfactant pool and increased intracellular pool. We originally hypothesized that after LPS administration there was increased clearance of surfactant phospholipids by type II cells. However, data from this study demonstrated that in vivo, surfactant clearance by type II cells was similar at both 18 and 72 hours after intrapulmonary LPS instillation compared to control groups killed at the same time points (Figs 4 and 5). Data from the current study cannot lead to any conclusions pertaining to alterations in type II cell phospholipid synthesis or secretion. As mentioned, we observed a small but not statistically significant decrease in alveolar phospholipid pools after LPS administration, which is in contrast to previous studies by Viviano et al. and MacIntosh et al. that used 1 mg/kg and 0.5 mg/kg of LPS respectively [9,10]. Nevertheless, we did observe a similar increase in alveolar levels of SP-A after intrapulmonary LPS (Fig 1), which is a well documented characteristic of lung inflammation after LPS exposure [9-11]. This increase has been primarily attributed to increased SP-A synthesis by type II cells [11] suggesting that indeed type II cell metabolism was altered in our model. Additionally, these data provide further experimental evidence that surfactant phospholipid and SP-A pools can be independently regulated in response to inflammatory agents. We also investigated additional tissue-associated cells that were isolated after lung digestion (primarily macrophages and neutrophils). Similar to type II cells, surfactant clearance from these tissue-associated cells isolated from the two LPS groups were similar to their respective control groups. In addition, the clearance attributed to type II cells and tissue-associated cells was similar at both time points for the LPS and control groups. This observation is in agreement with a study by Gurel et al., that documented that alveolar type II cells and tissue macrophages contributed equally to the alveolar clearance of phospholipid in adult mice [27]. Conclusions In conclusion, we demonstrated that after intrapulmonary LPS administration in adult rats, the in vivo clearance of surfactant lipids by type II cells is unaltered and that the increased clearance of surfactant lipids is primarily due to either neutrophils or macrophages that are recruited to the alveolar space. Authors' contributions JLM conceived, designed and performed all aspects of the study, and was the primary participant in its writing. JRW aided in conception and design of study, and participated in its writing. Acknowledgements JLM is a Parker B Francis Fellow in pulmonary research and was a recipient of a Post-Doctoral Fellowship from the Canadian Lung Association and Canadian Institutes of Health Research. Study was funded by RO1 HL-30923 (JRW). ==== Refs Goerke J Pulmonary surfactant: functions and molecular composition Biochim Biophys Acta 1998 1408 79 89 9813251 10.1016/S0925-4439(98)00060-X Crouch E Wright JR Surfactant proteins a and d and pulmonary host defense Annu Rev Physiol 2001 63 521 554 11181966 10.1146/annurev.physiol.63.1.521 Gregory TJ Longmore WJ Moxley MA Whitsett JA Reed CR Fowler AA Maunder RJ Crim C Hyers TM Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome J Clin Invest 1991 88 1976 1981 1752956 Pison U Obertacke U Brand M Seeger W Joka T Bruch J Schmit Neuerburg KP Altered pulmonary surfactant in uncomplicated and septicemia- complicated courses of acute respiratory failure J Trauma 1990 30 19 26 2296063 Gunther A Siebert C Schmidt R Ziegler S Grimminger F Yabut M Temmesfeld B Walmrath D Morr H Seeger W Surfactant alterations in severe pneumonia, acute respiratory distress syndrome, and cardiogenic lung edema Am J Respir Crit Care Med 1996 153 176 184 8542113 Vanderzwan J McCaig L Mehta S Joseph M Whitsett J McCormack DG Lewis JF Characterizing alterations in the pulmonary surfactant system in a rat model of Pseudomonas aeruginosa pneumonia Eur Respir J 1998 12 1388 1396 9877497 10.1183/09031936.98.12061388 Holm BA Notter RH Siegle J Matalon S Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia J Appl Physiol 1985 59 1402 1409 3840800 Bailey TC Cavanagh C Mehta S Lewis JF Veldhuizen RA Sepsis and hyperoxia effects on the pulmonary surfactant system in wild-type and iNOS knockout mice Eur Respir J 2002 20 177 182 12166567 10.1183/09031936.02.00276702 McIntosh JC Swyers AH Fisher JH Wright JR Surfactant proteins A and D increase in response to intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol 1996 15 509 519 8879185 Viviano CJ Bakewell WE Dixon D Dethloff LA Hook GE Altered regulation of surfactant phospholipid and protein A during acute pulmonary inflammation Biochim Biophys Acta 1995 1259 235 244 8541330 10.1016/0005-2760(95)00167-0 Sugahara K Iyama K Sano K Kuroki Y Akino T Matsumoto M Overexpression of surfactant protein SP-A, SP-B, and SP-C mRNA in rat lungs with lipopolysaccharide-induced injury Lab Invest 1996 74 209 220 8569184 Lewis JF Ikegami M Jobe AH Altered surfactant function and metabolism in rabbits with acute lung injury J Appl Physiol 1990 69 2303 2310 2077029 Malloy J McCaig L Veldhuizen R Yao L-J Joseph M Whitsett J Lewis J Alterations of the endogenous surfactant system in septic adult rats Am J Respir Crit Care Med 1997 156 617 623 9279248 Wright JR Dobbs LG Regulation of pulmonary surfactant secretion and clearance Annu Rev Physiol 1991 53 395 414 2042966 10.1146/annurev.ph.53.030191.002143 Wright JR Clearance and recycling of pulmonary surfactant Am J Physiol 1990 259 L1 12 2200279 Quintero OA Wright JR Clearance of surfactant lipids by neutrophils and macrophages isolated from the acutely inflamed lung Am J Physiol Lung Cell Mol Physiol 2002 282 L330 L339 11792638 Uhlig S Brasch F Wollin L Fehrenbach H Richter J Wendel A Functional and fine structural changes in isolated rat lungs challenged with endotoxin ex vivo and in vitro Am J Pathol 1995 146 1235 1247 7747816 Fehrenbach H Brasch F Uhlig S Weisser M Stamme C Wendel A Richter J Early alterations in intracellular and alveolar surfactant of the rat lung in response to endotoxin Am J Respir Crit Care Med 1998 157 1630 1639 9603148 Bligh EG Dyer WJ A rapid method of total lipid extraction and purification Can J Med Sci 1959 37 911 917 13671378 Duck-Chong CG A rapid sensitive method for determining phospholipid phosphorus involving digestion with magnesium nitrate Lipids 1979 14 492 497 Tenner AJ Robinson SL Borchelt J Wright JR Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1-mediated phagocytosis J Biol Chem 1989 264 13923 13928 2788165 Dobbs LG Isolation and culture of alveolar type II cells Am J Physiol 1990 258 L134 L147 2185652 Herbein JF Wright JR Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung Am J Physiol Lung Cell Mol Physiol 2001 281 L268 L277 11404270 Hartshorn KL Crouch E White MR Colamussi ML Kakkanatt A Tauber B Shepherd V Sastry KN Pulmonary surfactant proteins A and D enhance neutrophil uptake of bacteria Am J Physiol 1998 274 L958 L969 9609735 Rider ED Ikegami M Jobe AH Localization of alveolar surfactant clearance in rabbit lung cells Am J Physiol 1992 263 L201 L209 1514645 Lopez A Albassam M Yong S Sharma A Lillie LE Prior MG Profiles of type-II pneumocytes in rats inoculated intratracheally with bacterial lipopolysaccharide Am J Vet Res 1987 48 1534 1539 3314609 Gurel O Ikegami M Chroneos ZC Jobe AH Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs Am J Physiol Lung Cell Mol Physiol 2001 280 L1266 L1272 11350807

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==== Front Respir ResRespiratory Research1465-99211465-993XBioMed Central 1465-9921-5-91535788110.1186/1465-9921-5-9ResearchAdaptation of an amphibian mucociliary clearance model to evaluate early effects of tobacco smoke exposure Zayas J Gustavo [email protected]'Brien Darryl W [email protected] Shusheng [email protected] Jie [email protected] Leonard [email protected] Malcolm [email protected] Mucobiology Research Unit, Pulmonary Research Group, University of Alberta, Edmonton, Canada2004 20 8 2004 5 1 9 9 9 6 2003 20 8 2004 Copyright © 2004 Zayas et al; licensee BioMed Central Ltd.2004Zayas et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rationale Inhaled side-stream tobacco smoke brings in all of its harmful components impairing mechanisms that protect the airways and lungs. Chronic respiratory health consequences are a complex multi-step silent process. By the time clinical manifestations require medical attention, several structural and functional changes have already occurred. The respiratory system has to undergo an iterative process of injury, healing and remodeling with every exposure. Methods To have a better understanding of the initial changes that take place when first exposed to environmental tobacco smoke, we have developed an exposure model, using the frog palate that closely represents the features of obstructive airways where ciliary dysfunction and mucus hypersecretion occur. Results Mucus transport was significantly reduced, even after exposure to the smoke of one cigarette (p < 0.05) and even further with 4-cigarettes exposure (p < 0.001). Morphometric and ultrastructural studies by SEM show extensive areas of tissue disruption. Gelatinase zymography shows activation of MMP9 in mucus from palates exposed to tobacco smoke. Conclusions The clearance of mucus on the frog palate is significantly reduced after exposure to environmental tobacco smoke. Cilia and the extracellular matrix are anatomically disrupted. Tobacco smoke triggers an increased activity of matrix metalloproteinases associated with a substantial defoliation of ciliated epithelium. These studies enhance the knowledge of the changes in the mucociliary apparatus that occur initially after exposure to environmental tobacco smoke, with the goal of understanding how these changes relate to the genesis of chronic airway pathologies in humans. ==== Body Background Respiratory diseases, infectious and non-infectious, are a prime cause of morbidity, mortality and health system utilization in many countries. Exposure to cigarette smoke is an important factor in causing as well as increasing complications in several pulmonary disorders. The mucociliary clearance constitutes the first line of defense to maintain the airways as free as possible of foreign bodies [1]. Impairment of mucociliary function may be the result of epithelial airway damage, ciliary dysfunction, inflammation, and change in mucus viscosity and/or elasticity. In laboratory studies we have shown that the physical properties of mucus in nonsmokers are not altered by age or by restrictive pulmonary pathology. However, mucus properties show alterations when exposed to tobacco smoke and these alterations are noticeable in the very early stages of smoke exposure, even at an exposure level in the range of 1 to 5 cigarettes per day [2]. The majority of studies on the outcome of tobacco use have been performed in the late phases of the process [3-7]; the acute and early effects on cilia, mucus and mucociliary clearance after active smoking or side stream tobacco smoke exposure have not been well studied. We hypothesized that studying changes occurring in the initial stages will lead to a better understanding of the multifaceted problems of tobacco exposure. Therefore our laboratory has been developing and using in vivo and ex vivo epithelial injury models that replicate the features of airway diseases [8-12]. The mucus blanket in the frog palate is cleared by coordinated ciliary activity in an almost identical fashion to that observed in human airways. We anticipated that a modified frog palate exposure model could better serve our purposes. We were specifically interested in developing an exposure model that would allow us to study the initial effects and mechanisms occurring in an epithelial tissue after exposure to environmental tobacco smoke. We conceived this model to be free of interferences from other agents or systemic physiological responses resulting from other internal or external influences. We also aim to have a better understanding of the mechanism by which ciliated epithelial cells are exfoliated after being exposed to tobacco smoke, as this may relate directly to impaired mucus clearance in several human airway diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Hence we decided first to test our novel ex vivo model, and in later studies use in vivo models. Methodology Frog palate preparation From a bullfrog, Rana catesbiana, the upper portion of the head is removed following the procedures described in previous work [10,11], by cutting with scissors through from the junction of the posterior pharynx and esophagus out to the skin of the back. This procedure was carried out after lowering the body temperature of the frog for 30 – 60 minutes inside a refrigerator to abolish pain sensations. The palate was examined for macroscopic lesions, such as ulcers, petechia or redness as evidence of inflammation. Only palates free of inflammatory indicators were included in this study. Any blood remaining in the epithelial surface was carefully washed away, then the excised head was placed palate side facing upwards on a piece of gauze saturated with frog Ringer solution (FRS) in a Petri dish. The experimental procedures involving animals were approved by the Health Sciences Animal Policy and Welfare Committee, University of Alberta. The FRS was prepared by mixing standard Ringer injection with sterile water (2:1). The composition of standard frog Ringer (in mmol/l) is 90 NaCl, 3 KCl, 2 CaCl2, and 15 NaHCO3 (220 mosm/l). The palate was placed inside the frog chamber, a wooden box with a glass top and fitted glass front, and manipulated trough glove openings. The humidity inside the box is maintained at 100% using an ultrasonic Pari nebulizer and the temperature is kept between 22° to 24°C by a rheostat-controlled, externally mounted light source. Before carrying out any measurement, the palate was allowed to stabilize inside the box for 15 minutes before testing. Exposure chamber The exposure chamber (Figure 1) with a volume ~10 liters had two inlets: one connected to an ultrasonic Pari jet nebulizer system set at 8 L/min measured by a Puritan flow meter to maintain the chamber near 100 % humidity. The other inlet was linked to a burning chamber. The latter, which contained a burning cigarette, was slightly pressurized with air flowing into the chamber at a rate of 2 L/min to promote cigarette combustion. Positive ventilation inside the burning chamber pushed the side stream smoke into the covered but not sealed exposure chamber, which was exhausted into a fume hood. Temperature inside the chamber was maintained to 22°C. and monitored through a thermocouple and digital-display thermometer. The palate was placed inside with the palate side upwards on a piece of gauze saturated with FRS in a glass dish at about five centimeters above the bottom of the chamber. Figure 1 Box designed to expose the frog palate to cigarette smoke. The exposure chamber was designed to allow a steady flow of side stream tobacco smoke to reach the epithelial tissue. The concentration of cigarette smoke inside the exposure chamber was maintained constant throughout the experiment by the presence of a mixing baffle on the outflow of the chamber. The concentration of tobacco smoke inside the chamber was not directly measured, but is estimated that the half palate was exposed to the smoke of each cigarette delivered, diluted in 180 liters of fresh air. As much as possible, the conditions (humidity, type of solution, temperature, physical manipulation and airflow in and out) in the frog chamber and in the exposure chamber were maintained similar, with cigarette smoke being the independent variable. Frog palate exposure model preparation The palate was divided longitudinally in two halves along the midline as shown in Figure 2, cutting the epithelium with a scalpel to minimize damage. After five minutes the mucus transport was measured in both halves, left and right, to confirm that both half palates were functioning normally. One side was used as control and the other half was exposed to tobacco smoke. The control half of the palate was left in the frog chamber while the other half was placed in the exposure chamber maintained at similar conditions of 100 % humidity and room temperature. If any or both halves showed a variation in mucus transport greater than 30 % from the baseline, the experiment was aborted. Figure 2 The frog palate was further divided longitudinally in two halves following the midline. Mucus transport velocity (MTV) determination The palate was placed under a dissecting stereomicroscope provided with a reticulated eyepiece. Mucociliary clearance was determined by observing the movement of particles of charcoal powder gently deposited on a sample of mucus on the palate surface; its clearance was visually monitored and MTV determined. The displacement of 3 – 5 μL of endogenous frog mucus sample was calculated by dividing the distance traveled by the transit time across the 0.3 inch (7.62 mm) segment marked between 0.1 and 0.4 inches in the graduated eyepiece. At least five measurements of the time required for the mucus sample to travel the defined distance were made every time to obtain control and smoke exposure mucus transport velocity. We used cigarettes regularly available at commercial outlets of a representative brand in terms of customer preference and toxic emissions: nicotine 0.5 – 2.1 mg, tar 4 – 24 mg, carbon monoxide 5 – 25 mg, hydrogen cyanide 0.04 – 0.21 mg, benzene 0.025 – 0.069 mg, formaldehyde 0.018 – 0.1 mg. One cigarette took 17 minutes on average to burn completely in this preparation. Measurements and mucus samples collection After being exposed to one cigarette, the half palate was brought back to the frog chamber and MTV was measured in both control and exposed halves. A sample of mucus was collected from each half, and placed in separate containers and frozen at -80°C in liquid nitrogen followed by storage in a -80° freezer until analysis. The exposed half, brought back to the exposure chamber was further exposed to 3 more cigarettes (51 additional minutes of exposure). Following tobacco exposure, the half palate was again brought back to the frog chamber to measure mucus transport and collect tissue and mucus sample in both halves, control and exposed. One set of half palates (control and exposed to four cigarettes) was stored at 4°C overnight, and transport of mucus was reassessed the next day. Tissue and mucus samples collection Sample of mucus from both halves were immersed in glutaraldehyde 2.5%, and placed in properly identified separate containers for storage at 4°C for later Scanning Electron Microscope (SEM) studies. The epithelial tissue was carefully dissected and separated from the palate musculature. Samples of the palate tissue from each half were sectioned, frozen in liquid nitrogen and stored at -80°C for gelatinase zymography studies. Zymography Samples of tissue, as well as mucus, were taken out of the freezer and ground to a powder in a mortar and pestal. Liquid nitrogen was added to keep the samples frozen. Homogenizing buffer (KCl 100 mM, ZnCl2 0.5 mM, EDTA 10 mM, Tris-HCl 1 M, pH 6.8) was added to the ground samples (approximately 500 ul buffer per 10 mg tissue sample, 200 buffer per 10 mg mucus sample) that were sonicated for 30 seconds on ice and then centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was collected for protein assay (BCA protein assay kit, Pierce). Normalizing the protein content as 5 μg, different amount of samples were loaded into the 7.5% separating zymography gel (30% acrylamide/ 0.8% bisacrylamide 3.75 ml, 4 × Tris-Cl/ SDS, pH 8.8 3.75 ml, H2O 6 ml, 2% gelatin A 1.5 ml, 10% ammonium persulfate 0.1 ml, TEMED 0.01 ml, for 4 gels) and were run at 100 volts for 20 minutes, 150 volts for 40 minutes. After electrophoresis, the gel was washed 3 times (20 minutes per a time) in 2.5% Triton X-100 at room temperature followed by incubation for 96 hours in zymography development buffer (0.15 M NaCl, 5 mM CaCl2, 0.05% Azide NaN3, 50 mM Tris-HCl pH 7.6). The gel was then stained for 2 hours with stain solution (Coomassie brilliant blue R-250 1 g/L, methanol: acetic acid: H2O= 2.5: 1: 6.5) followed by de-staining (ethanol: acetic acid: H2O= 1: 2: 22) overnight. The expression of the gelatinases was shown on the gel as clear bands against the dark background stained with Coomassie blue. The bands were compared with the gelatinase standards for MMP 2 and 9 running in the first lane of each gel. The density of the gels was measured in the Bio-Rad scanning densitometer. Scanning electron microscopy Samples of mucus and tissue were placed in 2.5 % glutaraldehyde solution immediately after collection and stored at 4°C until processing. The samples were post-fixed in 1 % osmium tetraoxide in Milonig's buffer at room temperature for one hour. They were then washed in a series of ethanol (50 – 100 %), ten minutes at each step, followed by two additional periods of absolute ethanol (10 minutes each). The samples were further dehydrated by critical point drying at 31°C for 5 – 10 minutes, then mounted on a specimen holder for SEM and dried overnight in vacuum desiccators. In the final stage of preparation for viewing, the samples were sputter coated with gold (Edwards, model S150B Sputter Coater). Samples were viewed using SEM (Hitachi S-2500). Images were scanned directly to a computer and stored as image files for subsequent viewing and analysis. Statistical analysis Data are expressed as mean ± standard deviation unless otherwise stated. A paired Student-T test was used for simple comparison. The level of significance was set at 5 %. Results The modified fresh frog palate exposure model was relatively easy to prepare and practical to handle. On gross examination, the surface of the palate exposed to side stream tobacco smoke did not show any macroscopic change in appearance after cigarette smoke exposure (CSE) compared to the control halves maintained in the normal chamber. One half of the palate was used as a control and the other half was exposed to cigarette smoke. Baseline mucus transport velocity(MTV) was measured in both half palates prior to exposure of one half palate to cigarette smoke, and were identical (19.5 ± .03 mm/sec). Two additional MTV determinations were carried out in the control half after one and four cigarettes and after 24 h period of recovery during which time the palate was kept at 4°C. A paired T-test showed no statistical difference in mucus transport velocity among the control measurements during the entire experiment (Figure 3). Figure 3 Three MTV controls are shown in the graph, no differences were observed among them. After immediate exposure to four cigarettes, mucus transport on the palates was drastically reduced. One of the four cigarette-exposed palate and its respective half palate control were maintained overnight in a refrigerator, and it was not possible to measure a transport time the next day in the exposed palate. A paired T-test of MTV on the control half compared to the exposed half showed that mucus transport velocity in the exposed half palate was reduced (p < 0.03) immediately after exposure to the side stream smoke of one cigarette compared with the non-exposed half palate. Further exposure to the side stream smoke of three more cigarettes (4 in total) significantly reduced MTV (p < 0.001), with no signs of recovery after 24 hours. We obtained a sample of mucus from the half palate exposed to four cigarettes and used it on the control half palate to measure MTV. Clearance was within the normal range. Immediately after, the same sample of mucus was tested on the exposed half palate and mucus clearance was again seen to be very slow. Mucus transport had basically ceased in some areas of the tobacco-smoke exposed palate. Scanning electron microscopy (SEM) studies to assess the integrity of the epithelium after exposure to side stream smoke of one cigarette showed areas where the layer of cilia looked disordered. However, we did not see loss of cilia or exfoliation of ciliated epithelial cells after careful examination under the SEM in lower and high power of the entire surface of the sample after this level of exposure. In Figure 4, SEM images from palates exposed to the smoke of four cigarettes showed greater epithelial tissue disruption (panels 2 and 3) compared to a control palate (panel 1). Large areas of deciliated cells were observed, as well as exfoliation of intact ciliated cells. Examples of exfoliated cells found on the surface of the epithelium in the representative SEMs are indicated with black arrows. Morphometric analysis of the area of cilia loss from 3 paired palates exposed to four cigarettes, evaluating 12 different areas randomly selected in each palate, showed cilia loss of 51 ± 14 % compared to < 2 % on control palates. Figure 4 Magnification is ×400. On the left, the surface of the normal non-exposed palate is shown with a continuous ciliary layer, punctuated with secretory gland openings. The middle and right micrographs show the surface of palates exposed to the smoke of four cigarettes. Gelatinase zymography showed increased activity of MMP-9 in mucus collected from the palates exposed to tobacco smoke of four cigarettes compared to mucus from control palates as shown in Figure 5. MMP2 activity was not different in mucus samples obtained from palates exposed, or not to cigarette smoke, but these results are inconclusive. Figure 5 A representative gelatinase zymogram on mucus collected from control and cigarette-exposed palates. In the left lane, standards for MMP 2 and 9 are shown. In the next two lanes MMP activity is shown in control mucus (two samples). The next two lanes show MMP activity in two aliquots of the mucus collected from palates exposed to the smoke of four cigarettes. Discussion Major findings in this study include: a) our amphibian mucociliary clearance model appears to be appropriate to study the early effects of tobacco smoke exposure; b) the clearance of mucus is significantly reduced after exposure to side stream tobacco smoke associated to dose response; c) ciliated cells are anatomically and physiologically acutely affected therefore drastically impairing mucus clearance. Acute irritation and inflammation of cilia due to exposure to tobacco smoke could explain this alteration; d) mucus seems to not be physiologically affected at this stage; and e) tobacco smoke triggers an increased activity of matrix metalloproteinases (in particular MMP9) associated with disruption of the extracellular matrix, most likely affecting cellular attachments to the basal membrane generating a substantial exfoliation of the ciliated cells. These findings might assist us in enhancing the knowledge of the changes in the mucociliary apparatus that occur early after exposure to environmental tobacco products, with the goal to understand how these changes relate to the development of chronic airway pathologies in humans. The frog palate has been used for several decades as a model to assess mucociliary clearance [13-17]. Different species of frogs have also been used, as well as a variety of study designs. Researchers had to deal with several sources of variability, making it difficult to standardize a widely acceptable model. There are disadvantages in our model such that it pertains to a non-mammalian species, in addition that the epithelial tissue is non-respiratory. However as a model it has advantages over some mammalian models like rodents in that the ciliated epithelium has a well developed mucus blanket that work in coordination with cilia similar to the human situation. This exposure model is an isolated system, theoretically free of interferences from other agents or systemic physiological responses resulting from other internal or external influences. In this injury model we randomly exposed either half palate to side stream tobacco smoke and used the opposite half palate as the control (internal control). Since there was no statistical difference in mucus transport between them, previously established in different sets of pairs of palates, one side was arbitrarily selected for control and the opposite half for exposure to tobacco smoke. This study follows an approach utilized previously by Zayas et al [7] that compared samples of mucus obtained from both mainstem bronchi in smokers and in nonsmokers. In assessing the mucus transport rate in both half palates before any experimental procedure and to insure that they functioned identically, we established baseline data for our experiment. This helped to reduce error variability due to sex, weight, age, as well as control for any seasonal effect on the mucus transport rate in the frog palates as demonstrated by Rubin et al [18]. Since the palate is excised from the frog we may assume that any observed effect or response is a local and direct effect of exposure to side stream tobacco smoke, which is an advantage of our model. From the data obtained, we would conclude that our frog palate exposure model is suitable to study the acute effects of second hand tobacco smoke. We may also conclude that clearability of mucus seems not to be altered at this stage, at this level of exposure and in this particular exposure preparation or design. Therefore, at this stage mucus transport seems not to be functionally affected for being cleared in a normal fashion by non-exposed cilia. However, after more prolonged tobacco exposure there will be modifications in mucus properties, as seen and measured in smoking dogs where after two months of tobacco exposure, the galactose content and the viscoelastic properties of mucus were observed to present alterations. After further exposure the viscosity and elasticity properties of mucus were reversed to quasi-normal levels, but galactose content did not [12]. Hence the model makes it very useful for differentiation of cilia-related effects versus mucus-related effects on mucociliary clearance after acute tobacco exposure. Mucus clearance is a function of cilia beat frequency and mucus viscoelastic properties. A change in mucus clearance may be due to one or the other component or both. In future studies, incorporating high speed digital imaging of the palate surface to determine cilia beat frequency will allow us to further differentiate between cilia related effects versus mucus related effects. Exposure to tobacco smoke with all its noxious agents and components will possibly allow us to separate and study in detail, the timing and appearance of different phases of the organism response. Our exposure model may allow us to individualize and study the inflammation phase resulting from the tobacco exposure. We can then focus on the injury phase occurring in the mucociliary system. Later we will explore mechanisms involved in the healing process. The remodeling of the ciliated epithelium following acute injury will be an important component of our studies and particularly how the remodeling is mediated. The time frame of the injury and recovery phases needs to be determined. Cilia do not work alone, but in association with other cilia to produce metachronal waves. Several metachronal waves may contribute to the propulsion of the mucus layer to flow over irregularities or non-ciliated areas. Our data indicates that acute tobacco exposure may have an initial and early irritation effect, possibly mediated by an unknown mechanism on the exposed palate that leads to inhibition of ciliary beat frequency or discoordination of metachronal waves. Such factors adversely affect clearance by imposing stasis or local eddies resulting in erratic clearance that may be the reason why scanning electron micrographs of palates after one cigarette showed cilia somewhat disordered, but not visibly disrupted. We have shown that tobacco smoke exposure interferes with mucociliary clearance. Sustained exposure may lead to loss of ciliated epithelium associated with activation of matrix metalloproteinases. Significant loss of cilia or ciliated epithelial cells results in disruption or even cessation of mucociliary clearance. Matrix metalloproteinases may be implicated in this injury through disruption of epithelial cell-to-cell or cell-to-basement membrane connections. Further clarification of the mechanisms involved will be undertaken in subsequent studies. Our exposure model can assess physiological, ultrastructural and molecular parameters in response to the initial deleterious effects of acute exposure to side stream tobacco smoke in an epithelial model homologous to the human airways. In these preliminary studies we did not attempt to mimic "human smoking conditions". However, our results show that MTV is affected even after exposure to one cigarette. Although concentration of smoke used in the present study is higher than likely encountered in typical environmental tobacco exposures, they are within an order of magnitude of those computed for exposure in poorly ventilated cars or homes. Future studies will try to replicate real conditions faced by non-smokers exposed to environmental tobacco smoke and to characterize the mediation and effectors of this acute injury. Acknowledgements This study was funded by the Alberta Lung Association and the Canadian Institutes of Health Research. The authors would like to thank Dr. Ming Chen for his expert assistance with the scanning electron microscopy. ==== Refs King M Role of mucus viscoelasticity in cough clearance Biorheology 1987 24 589 597 3502760 Zayas JG Man GCW King M Tracheal mucus rheology in patients undergoing diagnostic bronchoscopy: Interrelations with smoking and cancer Am Rev Respir Dis 1990 141 1107 1113 2339832 Dulfano MJ ed Charles C Thomas Sputum: Fundamentals and Clinical Pathology 1973 Springfield, Illinois King M Wight A DeSanctis GT ElAzab J Phillips DM Angus GE Cosio MG Mucus hypersecretion and viscoelastic changes in cigarette-smoking dogs Exp Lung Res 1989 15 375 389 2743951 Schultheis AH Bassett DJ Fryer AD Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function J Appl Physiol 1994 76 1088 1097 8005850 Romieu I Meneses F Sienra-Monge JJ Effects of urban air pollutants on emergency visits for childhood asthma in Mexico City Am J Epidemiol 1995 141 546 53 7900722 Zayas JG Rubin BK York E Lien D King M Bronchial mucus properties in lung cancer. Relationship with site of lesion Can Respir J 1999 6 246 252 10393286 King M Wight A DeSanctis GT ElAzab J Phillips DM Angus GE Cosio MG Mucus hypersecretion and viscoelastic changes in cigarette-smoking dogs Exp Lung Res 1989 15 375 389 2743951 King M Experimental models for studying mucociliary clearance Eur Respir J 1998 11 222 228 9543296 10.1183/09031936.98.11010222 King M Festa E Baum GL Evolution of the frog palate model for mucociliary clearance 1970 – 1997 In Cilia, Mucus, and Mucociliary Interactions 1998 New York: Marcel Dekker 191 201 Rubin BK Ramirez O King M Mucus-depleted frog palate as a model for the study of mucociliary clearance J Appl Physiol 1990 69 2 424 29 2228850 Tai S Sun F O'Brien DW Lee MS Zayas JG King M Evaluation of a mucoactive herbal drug, Radix Ophiopogonis, in a pathogenic quail model Journal of Herbal Pharmacotherapy 2002 2 49 56 15277081 10.1300/J157v02n04_05 Miani P Piragine F Studi sugli epiteli vibratile delle prima vie aeree. I. Metodi in uso per lo studio del movimento ciliare Clinica ORL 1962 3–4 143 171 Suzuki N Motor control of the ciliary activity in the frog palate J Fac Sci Hokkaido Univ Sez VI Zoology 1966 16 67 71 Sade J Eliezer N Silberberg A Nevo AC The role of mucus in transport by cilia Am Rev Respir Dis 1970 102 48 52 5427403 King M Gilboa A Meyer FA Silberberg A On the transport of mucus and its rheologic stimulants in ciliated systems Am Rev Respir Dis 1974 110 740 744 4429269 Puchelle E Zahm JM Sadoul P Mucociliary frequency of frog palate epithelium Am J Physiol 1982 242 C31 C35 6977273 Rubin BK Cheeseman CI Gourishankar S King M Is there a seasonal variation in mucus transport and nutrient absorption in the leopard frog? Can J Physiol Pharmacol 1992 70 442 46 1498713

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1131531895110.1186/1471-2105-5-113SoftwareMUSCLE: a multiple sequence alignment method with reduced time and space complexity Edgar Robert C [email protected] Department of Plant and Microbial Biology, 461 Koshland Hall, University of California, Berkeley, CA 94720-3102, USA2004 19 8 2004 5 113 113 25 3 2004 19 8 2004 Copyright © 2004 Edgar; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In a previous paper, we introduced MUSCLE, a new program for creating multiple alignments of protein sequences, giving a brief summary of the algorithm and showing MUSCLE to achieve the highest scores reported to date on four alignment accuracy benchmarks. Here we present a more complete discussion of the algorithm, describing several previously unpublished techniques that improve biological accuracy and / or computational complexity. We introduce a new option, MUSCLE-fast, designed for high-throughput applications. We also describe a new protocol for evaluating objective functions that align two profiles. Results We compare the speed and accuracy of MUSCLE with CLUSTALW, Progressive POA and the MAFFT script FFTNS1, the fastest previously published program known to the author. Accuracy is measured using four benchmarks: BAliBASE, PREFAB, SABmark and SMART. We test three variants that offer highest accuracy (MUSCLE with default settings), highest speed (MUSCLE-fast), and a carefully chosen compromise between the two (MUSCLE-prog). We find MUSCLE-fast to be the fastest algorithm on all test sets, achieving average alignment accuracy similar to CLUSTALW in times that are typically two to three orders of magnitude less. MUSCLE-fast is able to align 1,000 sequences of average length 282 in 21 seconds on a current desktop computer. Conclusions MUSCLE offers a range of options that provide improved speed and / or alignment accuracy compared with currently available programs. MUSCLE is freely available at . ==== Body Background Multiple alignments of protein sequences are important in many applications, including phylogenetic tree estimation, secondary structure prediction and critical residue identification. Many multiple sequence alignment (MSA) algorithms have been proposed; for a recent review, see [1]. Two attributes of MSA programs are of primary importance to the user: biological accuracy and computational complexity (i.e., time and memory requirements). Complexity is of increasing relevance due to the rapid growth of sequence databases, which now contain enough representatives of larger protein families to exceed the capacity of most current programs. Obtaining biologically accurate alignments is also a challenge, as the best methods sometimes fail to align readily apparent conserved motifs [2]. We recently introduced MUSCLE, a new MSA program that provides significant improvements in both accuracy and speed, giving only a summary of the algorithm [2]. Here, we describe the MUSCLE algorithm more fully and analyze its complexity. We introduce a new option designed for high-throughput applications, MUSCLE-fast. We also describe a new method for evaluating objective functions for profile-profile alignment, the iterated step in the MUSCLE algorithm. Current methods While multiple alignment and phylogenetic tree reconstruction have traditionally been considered separately, the most natural formulation of the computational problem is to define a model of sequence evolution that assigns probabilities to all possible elementary sequence edits and then to seek an optimal directed graph in which edges represents edits and terminal nodes are the observed sequences. This graph makes the history explicit (it can be interpreted as a phylogenetic tree) and implies an alignment. No tractable method for finding an optimal graph is known for biologically realistic models, and simplification is therefore required. A common heuristic is to seek a multiple alignment that maximizes the SP score (the summed alignment score of each sequence pair), which is NP complete [3]. It can be achieved by dynamic programming with time and space complexity O(LN) in the sequence length L and number of sequences N [4], and is practical only for very small N. Stochastic methods such as Gibbs sampling can be used to search for a maximum objective score [5], but have not been widely adopted. A more popular strategy is the progressive method [6,7] (Figure 1), which first estimates a phylogenetic tree. A profile (a multiple alignment treated as a sequence by regarding each column as a symbol) is then constructed for each node in the binary tree. If the node is a leaf, the profile is the corresponding sequence; otherwise its profile is produced by a pair-wise alignment of the profiles of its child nodes (Figure 2). Current progressive algorithms are typically practical for up to a few hundred sequences on desktop computers, the best-known of which is CLUSTALW [8]. A variant of the progressive approach is used by T-Coffee [9], which builds a library of both local and global alignments of every pair of sequences and uses a library-based score for aligning two profiles. On the BAliBASE benchmark [10,11], T-Coffee achieves the best results reported prior to MUSCLE, but has a high time and space complexity that limits the number of sequences it can align to typically around one hundred. In our experience, errors in progressive alignments can often be attributed to one of the following issues: sub-optimal branching order in the tree, scoring parameters that are not optimal for a particular set of sequences (especially gap penalties), and inappropriate boundary conditions (e.g., seeking a global alignment of proteins having different domain organizations). Misalignments are sometimes readily apparent, motivating further processing (refinement). One approach is to use a progressive alignment as the initial state of a stochastic search for a maximum objective score (stochastic refinement). Alternatively, pairs of profiles can be extracted from the progressive alignment and re-aligned, keeping the results only when an objective score is improved (horizontal refinement) [12]. Implementation The basic strategy used by MUSCLE is similar to that used by PRRP [13] and MAFFT [14]. A progressive alignment is built, to which horizontal refinement is then applied. Algorithm overview MUSCLE has three stages. At the completion of each stage, a multiple alignment is available and the algorithm can be terminated. Stage 1: draft progressive The first stage builds a progressive alignment. Similarity measure The similarity of each pair of sequences is computed, either using k-mer counting or by constructing a global alignment of the pair and determining the fractional identity. Distance estimate A triangular distance matrix is computed from the pair-wise similarities. Tree construction A tree is constructed from the distance matrix using UPGMA or neighbor-joining, and a root is identified. Progressive alignment A progressive alignment is built by following the branching order of the tree, yielding a multiple alignment of all input sequences at the root. Stage 2: improved progressive The second stage attempts to improve the tree and builds a new progressive alignment according to this tree. This stage may be iterated. Similarity measure The similarity of each pair of sequences is computed using fractional identity computed from their mutual alignment in the current multiple alignment. Tree construction A tree is constructed by computing a Kimura distance matrix and applying a clustering method to this matrix. Tree comparison The previous and new trees are compared, identifying the set of internal nodes for which the branching order has changed. If Stage 2 has executed more than once, and the number of changed nodes has not decreased, the process of improving the tree is considered to have converged and iteration terminates. Progressive alignment A new progressive alignment is built. The existing alignment is retained of each subtree for which the branching order is unchanged; new alignments are created for the (possibly empty) set of changed nodes. When the alignment at the root is completed, the algorithm may terminate, return to step 2.1 or go to Stage 3. Stage 3: refinement The third stage performs iterative refinement using a variant of tree-dependent restricted partitioning [12]. Choice of bipartition An edge is deleted from the tree, dividing the sequences into two disjoint subsets (a bipartition). Edges are visiting in order of decreasing distance from the root. Profile extraction The profile (multiple alignment) of each subset is extracted from the current multiple alignment. Columns containing no residues (i.e., indels only) are discarded. Re-alignment The two profiles obtained in step 3.2 are re-aligned to each other using profile-profile alignment. Accept/reject The SP score of the multiple alignment implied by the new profile-profile alignment is computed. If the score increases, the new alignment is retained, otherwise it is discarded. If all edges have been visited without a change being retained, or if a user-defined maximum number of iterations has been reached, the algorithm is terminated, otherwise it returns to step 3.1. Visiting edges in order of decreasing distance from the root has the effect of first re-aligning individual sequences, then closely related groups Algorithm elements In the following, we describe the elements of the MUSCLE algorithm. In several cases, alternative versions of these elements were implemented in order to investigate their relative performance and to offer different trade-offs between accuracy, speed and memory use. Most of these alternatives are made available to the user via command-line options. Four benchmark datasets have been used to evaluate options and parameters in MUSCLE: BAliBASE [10,11], SABmark [15], SMART [16-18] and our own benchmark, PREFAB [2]. Objective score In its refinement stage, MUSCLE seeks to maximize an objective score, i.e. a function that maps a multiple sequence alignment to a real number which is designed to give larger values to better alignments. MUSCLE uses the sum-of-pairs (SP) score, defined to be the sum over pairs of sequences of their alignment scores. The alignment score of a pair of sequences is computed as the sum of substitution matrix scores for each aligned pair of residues, plus gap penalties. Gaps require special consideration (Figure 3). We use the term indel for the symbol that indicates a gap in a column (typically a dash '-'), reserving the term gap for a maximal contiguous series of indels. The gap penalty contribution to SP for a pair of sequences is computed by discarding all columns in which both sequences have an indel, then applying an affine penalty g + λe for each remaining gap where g is the per-gap penalty, λ is the gap length (number of indels in the gap), and e is the gap-length penalty (sometimes called the extension penalty). Progressive alignment Progressive alignment requires a rooted binary tree in which each sequence is assigned to a leaf. The tree is created by clustering a triangular matrix containing a distance measure for each pair of sequences. The branching order of the tree is followed in postfix order (i.e., children are visited before their parent). At each internal node, profile-profile alignment is used to align the existing alignments of the two child subtrees, and the new alignment is assigned to that node. A multiple alignment of all input sequences is produced at the root node (Figure 1). Similarity measures We use the term similarity for a measure on a pair of sequences that indicates their degree of evolutionary divergence (the sequences are assumed to be related). MUSCLE uses two types of similarity measure: the fractional identity D computed from a global alignment of the two sequences, and measures obtained by k-mer counting. A k-mer is a contiguous subsequence of length k, also known as a word or k-tuple. Related sequences tend to have more k-mers in common than expected by chance, provided that k is not too large and the divergence is not too great. Many sequence comparison methods based on k-mer counting have been proposed in the literature; for a review, see [19]. The primary motivation for these measures is improved speed as no alignment is required. MAFFT uses k-mer counting in a compressed alphabet (i.e., an alphabet in which symbols denote classes that may contain two or more residue types) to compute its initial distance measure. The alphabet used in MAFFT is taken from [20], and is one of the options implemented in MUSCLE. Trivially, identity is higher or equal in a compressed alphabet; it cannot be reduced. If the alphabet is chosen such that there are high probabilities of intra-class substitution and low probabilities of inter-class substitution, then we might expect that detectable identity (and hence the number of conserved k-mers) could be usefully extended to greater evolutionary distances while limiting the increase in matches due to chance. We have previously shown [21] that k-mer similarities correlate well with fractional identity, although we failed to find evidence that compressed alphabets have superior performance to the standard alphabet at lower identities. We define the following similarity measure between sequences X and Y: F = Στ min [nX(τ), nY(τ) ] / [min (LX, LY) - k + 1 ].     (1) Here τ is a k-mer, LX, LY are the sequence lengths, and nX(τ) and nY(τ) are the number of times τ occurs in X and Y respectively. This definition can be motivated by considering an alignment of X to Y and defining the similarity to be the fraction of k-mers that are conserved between the two sequences. The denominator of F is the maximum number of k-mers that could be aligned. Note that if a given k-mer occurs more often in one sequence than the other, the excess cannot be conserved, hence the minimum in the numerator. The definition of F is an approximation in which it is assumed that (after correcting for excesses) common k-mers are always alignable to each other. MUSCLE also implements a binary approximation FBinary, so-called because it reduces the k-mer count to a present / absent bit: FBinary = Στ δXY(τ) / [min (LX, LY) - k + 1 ].     (2) Here, δXY(τ) is 1 if τ is present in both sequences, 0 otherwise. As multiple instances of a given k-mer in one sequence are relatively rare, this is often a good approximation to F. The binary approximation enables a significant speed improvement as the size of the count vector for a given sequence can be reduced by an order of magnitude. This allows the count vector for every sequence to be retained in memory, and pairs of vectors to be compared efficiently using bit-wise instructions. When using an integer count, there may be insufficient memory to store all count vectors, making it necessary to re-compute counts several times for a given sequence. Distance measures Given a similarity value, we wish to estimate an additive distance measure. An additive measure distance measure d(A, B) between two sequences A and B satisfies d(A, B) = d(A, C) + d(C, B) for any third sequence C, assuming that A, B and C are all related. Ideal but generally unknowable is the mutation distance, i.e. the number of mutations that occurred on the historical path between the sequences. The historical path through the phylogenetic tree extends from one sequence to the other via their most recent common ancestor. The mutation distance is trivially additive. The fractional identity D is often used as a similarity measure; for closely related sequences 1 - D is a good approximation to a mutation distance (it is exact assuming substitution at a single site to be the only allowed type of mutation and that no position mutates more than once). As sequences diverge, there is an increasing probability of multiple mutations at a single site. To correct for this, we use the following distance estimate [22]: dKimura = -loge (1 - D - D2/5)     (3) For D ≤ 0.25 we use a lookup table taken from the CLUSTALW source code. For k-mer measures, we use: dkmer = 1 - F.     (4) Tree construction Given a distance matrix, a binary tree is constructed by clustering. Two methods are implemented: neighbor-joining [23], and UPGMA [24]. MUSCLE implements three variants of UPGMA that differ in their assignment of distances to a new cluster. Consider two clusters (subtrees) L and R to be merged into a new cluster P, which becomes the parent of L and R in the binary tree. Average linkage assigns this distance to a third cluster C: dAvgPC = (dLC + dRC)/2.     (5) We can take the minimum rather than the average: dMinPC = min [dLC, dRC].     (6) Following MAFFT, we also implemented a weighted mixture of minimum and average linkage: dMixPC = (1 - s) dMinPC + s dAvgPC,     (7) where s is a parameter set to 0.1 by default. Clustering produces a pseudo-root (the last node created). We implemented two other methods for determining a root: minimizing the average branch weight [25], as used by CLUSTALW, and locating the root at the center of the longest span. Sequence weighting Conventional wisdom holds that sequences should be weighted to correct for the effects of biased sampling from a family of related proteins; however, there is no consensus on how such weights should be computed. MUSCLE implements the following sequence weighting schemes: none (all sequences have equal weight), Henikoff [26], PSI-BLAST [27] (a variant of Henikoff), CLUSTALW's, GSC [28], and the three-way method [29]. We found the use of weighting to give a small improvement in benchmark accuracy results, e.g. approximately 1% on BAliBASE, but saw little difference between the alternative schemes. The CLUSTALW method enables a significant reduction in complexity (described later), and is therefore the default choice. Profile functions In order to apply pair-wise alignment methods to profiles, a scoring function must be defined for a pair of profile positions, i.e. a pair of multiple alignment columns. This function is the profile analog of a substitution matrix; see for example [30]. We use the following notation. Let i and j be amino acid types, pi the background probability of i, pij the joint probability of i and j being aligned to each other, Sij the substitution matrix score, f xi the observed frequency of i in column x of the first profile, f xG the observed frequency of gaps in that column, and αxi the estimated probability of observing i in position x in the family. (Similarly for position y in the second profile). Estimated probabilities α are derived from the observed frequencies f, typically by adding heuristic pseudo-counts or by using Bayesian methods such as Dirichlet mixture priors [31]. A commonly used profile function is the sequence-weighted sum of substitution matrix scores for each pair of letters, selecting one from each column (PSP, for profile SP): PSPxy = Σi Σj f xi f yj Sij.     (8) Note that Sij = log (pij / pipj) [32], so PSPxy = Σi Σj f xi f yj log (pij / pi pj).     (9) PSP is the function used by CLUSTALW and MAFFT. It is a natural choice when attempting to maximize the SP objective score: if gap penalties are neglected, maximizing PSP maximizes SP under the constraint that columns in each profile are preserved. (This follows from the observation that the contribution to SP from a pair of sequences in the same profile is the same for all alignments allowed under the constraint). MUSCLE implements PSP functions based on the 200 PAM matrix of [33] and the 240 PAM VTML matrix [34]. In addition to PSP, MUSCLE implements a function we call the log-expectation (LE) score. LE is a modified version of the log-average (LA) profile function that was proposed on theoretical grounds [35]: LAxy = log Σi Σj αxi αyj pij / pi pj.     (10) LE is defined as follows: LExy = (1 - f xG) (1 - f yG) log Σi Σ j f xi f yj pij / pipj.     (11) The MUSCLE LE function uses probabilities computed from VTML 240. Note that estimated probabilities α in LA are replaced by observed frequencies f in LE. The factor (1 - fG) is the occupancy of a column. Frequencies fi must be normalized to sum to one if indels are present (otherwise the logarithm becomes increasingly negative with increasing numbers of gaps even when aligning conserved or similar residues). The occupancy factors are introduced to encourage more highly occupied columns (i.e., those with fewer gaps) to align, and are found to significantly improve accuracy. We avoid these complications in the PSP score by computing frequencies in a 21-letter alphabet (amino acids + indel), and by defining the substitution score of an amino acid to an indel to be zero. This has the desired effect of down-weighting column pairs with low occupancies, and can also be motivated by consideration of the SP function. If gap penalties are ignored, then this definition of PSP preserves the optimization of SP under the fixed-column constraint by correctly accounting for the reduced number of residue pairs in columns containing gaps. Gap penalties We call the first indel in a gap its gap-open; the last its gap-close. Consider an alignment of two profiles X and Y, and a gap of length λ in X in which the gap-open is aligned to position yo in Y and the gap-close to yc. The penalty for this gap is b(yo) + t(yc) + λe, where b and t are costs for opening and closing a gap that vary according to the position in Y, and e is a length cost (sometimes called a gap extension penalty) that does not vary by position. A fixed length cost allows a minor optimization of the scoring scheme [14]. Consider a global alignment of sequences X and Y having lengths LX and LY. If a constant δ (the center) is added to each substitution matrix score and δ/2 is added for each gapped position, this adds the constant value δ(LX + LY)/2 to the score of any possible alignment, and the set of optimal alignments is therefore unchanged. Given a scoring scheme with substitution matrix Sij and extension penalty e, we can thus choose δ/2 = e and instead use S'ij = Sij + 2e and e' = 0 to obtain the same alignment. The constant 2e can be added to the substitution matrix at compile time, and no explicit extension penalty is then needed in the recursion relations. MUSCLE uses this optimization for the PSP function, but not for LE (where the center must be added at execution time after taking the logarithm). Let f yo be the number of gap-opens in column y in Y and f yc be the number of gap-closes in column y. MUSCLE computes b and t as follows (Figure 4): b(y) = g/2 (1 - f yo) (1 + hw(y)H),     (12) t(y) = g/2 (1 - f yc) (1 + hw(y)H).     (13) Here, g is a parameter that can be considered a default per-gap penalty, hw(y) is 1 if y falls within a window of w consecutive hydrophobic residues or zero otherwise, and H is a tunable parameter. By default, w = 5, H = 1.2. The factor g/2 (1 - f yo) is motivated by considering the SP score of the alignment. The gap penalty contribution to SP for a pair of sequences (A ∈ Y, B ∈ X) is computed by discarding all columns in which both sequences have an indel, then applying an affine penalty g + λe for each remaining gap. It is convenient here to consider that half of the per-gap penalty g is applied to the open position and half to the close position. Suppose a gap G is inserted into X such that the gap-open is aligned to position y in Y. If a sequence s ∈ Y has a gap-open at y, then the SP score includes no open penalty for G induced by any pair (s, t) : t ∈ X. The multiplier (1 - f yo) therefore corrects the gap-open contribution to the SP score due to pre-existing gaps in Y. (It should be noted that even with this correction, there are other issues related to gaps and PSP still does not exactly optimize SP under the fixed-column constraint). The increased penalty in hydrophobic windows is designed to discourage gaps in buried core regions where insertions and deletions are less frequent. Note that MUSCLE treats open and close positions symmetrically, in contrast to CLUSTALW, which treats the open position specially and may therefore tend to produce, in word processing terms, left-aligned gaps with a ragged right margin. Terminal gaps A terminal gap is one that opens at the N-terminal position of the sequence to which it is aligned or closes at the C-terminal; as opposed to an internal gap. It has been suggested [9,36] that global methods have intrinsic difficulties with long deletions or insertions. We believe that these difficulties are often due to the choice of penalties for terminal gaps. CLUSTALW, which charges no penalty for terminal gaps, tends to fail to open a needed internal gap and thus fail to align terminal motifs; MAFFT, which charges the same penalty for terminal and internal gaps, sometimes aligns small numbers of residues to a terminal by inserting an unnatural internal gap. By default, MUSCLE penalizes terminal gaps with half the penalty of an internal gap. This is done by setting b(1), the open penalty at the C-terminal, and t(L), the close penalty at the N-terminal, to zero (Figure 4). The option of always applying full penalties, as in MAFFT, is also provided. We found that the compromise of a half penalty for terminal gaps gave good results for a wide range of input data, but that further improvements could sometimes by achieved by the following technique. If the length ratio of the two profiles to be aligned exceeds a threshold (by default, 20%), then MUSCLE constructs four different alignments in which gaps at both, one or neither terminals are fully penalized. A conservation score is defined by subtracting all gap penalties (both internal and terminal) from the alignment score, leaving a sum over profile functions only. The alignment with the highest conservation score is used. Tree comparison In progressive alignment, two subtrees will produce identical alignments if they have the same set of sequences at their leaves and the same branching orders (topologies). We exploit this observation to optimize the progressive alignment in Stage 2 of MUSCLE, which begins by constructing a new tree. Unchanged subtrees are identified, and their alignments are retained (Figure 5). A progressive alignment of the changed subtrees is constructed, producing the same alignment at the root that would be obtained starting from the leaves. Tree comparison is performed by the following algorithm. Consider two trees A and B with identical sets of N leaves. Leaves are identified by consecutive integers (ids) 1 through N. Call a pair of nodes, one from each tree, equivalent if they are the same leaf or they are internal nodes and their children are equivalent. The left/right position of a child is not considered; in other words, subtree rotations are allowed (because they do not change the results of a progressive alignment). Traverse A in prefix order (children before their parent), assigning internal nodes ids N + 1 through 2N in the order visited. When visiting an internal node PA, take the ids of its two child nodes LA and RA and use them as indexes into a lookup table pointing to nodes in B. If (a) LA is equivalent to a node LB in B and RA is equivalent to a node RB, and (b) LB and RB have the same parent PB, then assign PB the same id as PA, to which it is equivalent. When the traversal is complete, a node b in B is equivalent to some node in A if and only if b has an id. This procedure is O(N) time and space. Defaults, optimizations and complexity analysis We now discuss the default choices of algorithm elements in the MUSCLE program and analyze their complexity. Complexity of CLUSTALW It is instructive to consider the complexity of CLUSTALW. This is of intrinsic interest as CLUSTALW is currently the most widely used MSA program and, to the best of our knowledge, its complexity has not previously been stated correctly in the literature. It is also useful as a baseline for motivating some of the optimizations used in MUSCLE. The CLUSTALW algorithm can be described by the same steps as Stage 1 above. The similarity measure is the fractional identity computed from a global alignment, clustering is done by neighbor-joining. Global alignment of a pair of sequences or profiles is computed using the Myers-Miller linear space algorithm [37] which is O(L) space and O(L2) time in the typical sequence length L. Given N sequences and thus N(N - 1)/2 = O(N2) pairs, it is therefore O(N2L2) time and O(N2 + L) space to construct the distance matrix. The neighbor-joining implementation is O(N2) space and O(N4) time, at least up to CLUSTALW 1.82, although O(N3) time is possible; see e.g. [38]. A single iteration of progressive alignment computes a profile of each subtree from its multiple alignment, which is O(NPLP) time and space in the number of sequences in the profile NP and the profile length LP, then uses Myers-Miller to align the profiles in O(LP) space and O(LP2) time. There are N - 1 internal nodes in a rooted binary tree and hence O(N) iterations. It is often assumed that LP is O(L), i.e. that O(0) gaps are introduced in each iteration. However, we often observe the alignment length to grow approximately linearly, i.e. that O(1) gaps are added per iteration. For example, taking the average over all iterations in all alignments in version 3 of the PREFAB benchmark, Stage 1 of MUSCLE adds 2.8 gaps per iteration to the longer profile. It is therefore more realistic to assume that LP is O(L + N), making one iteration of progressive alignment O(NL + L2) in both space and time. This analysis is summarized in Table 1. Initial distance measure One might expect (a) that a more accurate distance measure would lead to a more accurate final alignment due to an improved tree, and (b) that errors due to a less accurate distance measure might be eliminated by allowing Stage 2 to iterate more times. Neither of these expectations is supported by our test results (unpublished). Allowing Stage 2 to iterate more than once with the goal of further improving the tree gave no significant improvement with any distance measure. Possibly, the tree is biased towards the MSA that was used to estimate it, and the MSA is biased by the tree used to create it, making it hard to achieve improvements. The most accurate measure on a pair of sequences is presumably the fractional identity D computed from a global alignment, but use of D in step 1.1 does not improve average accuracy on benchmark tests. The 6-class Dayhoff alphabet used by MAFFT proved to give slightly higher benchmark accuracy scores, despite the fact that other alphabets were found to correlate better with D [21]. We also found that the use of the binary approximation FBinary gave slightly reduced accuracy scores even when Stage 2 was allowed to iterate. The default choice in MUSCLE is therefore to use the Dayhoff alphabet in step 1.1 and to execute Stage 2 once only. While the impact on the average accuracy of the final alignment due to the different options is not understood, we observe that a better alignment of a pair of sequences is often obtained from a multiple alignment than from a pair-wise alignment, due to the presence of intermediate sequences having higher identities. It is therefore plausible that D obtained from the multiple alignment in step 2.1 may be more accurate than D obtained from a pair-wise alignment in step 1.1, and this may be relatively insensitive to the method used to create the tree for Stage 1. But this leaves unexplained why k-mer counting appears to be as good as or better than D in Stage 1. Computing F from a pair of sequences is O(L) time and O(1) space, so for all pairs the similarity calculation is O(N2L), compared with O(N2L2) in CLUSTALW. For a typical L around 250, combined with an order of magnitude improvement due to the simplicity of k-mer counting compared with dynamic programming, this typically gives a three orders of magnitude speed improvement for computing the distance matrix in MUSCLE compared with CLUSTALW. The default strategy is therefore well justified as a speed optimization, and has the added bonus of providing a small improvement in accuracy. Clustering MUSCLE implements both UPGMA and neighbor-joining. We found UPGMA to give slightly better benchmark scores than neighbor-joining; UPGMA is therefore the default option. We expect neighbor-joining to give a better estimate of the correct evolutionary tree (see e.g. [38]). However, it is well-known that alignment accuracy decreases with lower sequence identity (see e.g. [39]). It follows that given a set of profiles, the two that can be aligned most accurately will tend to be the pair with the highest identity, i.e. at the shortest evolutionary distance. This is exactly the pair selected by the nearest-neighbor criterion in UPGMA. By contrast, neighbor-joining selects a pair of evolutionary neighbors, i.e. a pair having a common ancestor. When mutation rates are variable, the evolutionary neighbor may not be the nearest neighbor (Figure 6). This explains why a nearest-neighbor tree may be superior to the true evolutionary tree for guiding a progressive alignment. Neighbor-joining is naively O(N4) time, although this can be reduced to O(N3). UPGMA is naively O(N3) time as the minimum of an N2 matrix must be found in each of N - 1 iterations. However, this can be reduced to O(N2) time by maintaining a vector of pointers to the minimum value in each row of the matrix. We are again fortunate to find that the most accurate method is also the fastest. Dynamic programming The textbook algorithm for pair-wise alignment with affine penalties employs three dynamic programming matrices; see e.g. [40,41]. A more time-and space-efficient implementation can be achieved using linear space for the recursion relations and a single matrix for trace-back (Kazutaka Katoh, personal communication). Consider sequences X and Y length LX, LY. We use the following notation: Xx is the xth letter in X, Xx the first x letters in X, Sxy the substitution score (or profile function) for aligning Xx to Yy, bXx the score for a gap-open in Y that is aligned to Xx, tXx the score for a gap-close aligned to Xx, Uxy the set of all alignments of Xx to Yy, Mxy the score of the best alignment in Uxy ending in a match (i.e., Xx and Yy are aligned), Dxy the score of the best alignment ending in a delete relative to X (Xx is aligned to an indel) and Ixy the score of the best alignment ending in an insert (Yy is aligned to an indel). A match is preceded by either a match, delete or insert, so: Mxy = Sxy + max { Mx-1y-1, Dx-1y-1 + tXx-1, Ix-1y-1 + tYy-1}     (14) We assume that a center parameter has been added to Sxy such that the gap extension penalty is zero. By considering all possible lengths for the final gap, Dxy = max(k<x) [Mky + bXk+1].     (15) Here, k is the last position in X that is aligned to a letter in Y. Extract the special case of a gap of length 1: Dxy = max { max(k<x-1) [Mky + bXk+1], Mx-1y + bXx}.     (16) Hence, Dxy = max { Dx-1y, Mx-1y + bXx }.     (17) Similarly, Ixy = max { Ixy-1, Mxy-1 + bYy }.     (18) Let the outer loop iterate over increasing x and the inner loop over increasing y. For fixed x, define vectors Mcurry = Mxy, Mprevy = Mx-1y, Dcurrx = Dxy, Dprevx = Dx-1y; for fixed x, y define scalars Icurr = Ixy, Iprev = Ixy-1. Now we can re-write (14), (17) and (18) to obtain the following recursion relations: Mcurry = Sxy + max { Mprevy-1, Dprev y-1 + tXx-1, Iprevy-1 + tYy-1 }     (19) Dcurry = max { Dprevy, Mprevy + bXx }     (20) Icurr = max { Iprev, Mprevy + bYy }.     (21) An LX × LY matrix is needed for the trace-back that produces the final alignment. Inner loop The inner-most dynamic programming loop, which computes the profile function, deserves careful optimization. We will consider the case of PSP; similar optimizations are possible for LE. PSP = Σi Σj f xi f yj Sij = Σi f xi Wyi, where Wyi = Σj f yj Sij. The vector Wyi is used LX times, and it therefore pays to compute it once and cache it. Observe that a typical profile column contains << 20 different amino acids. We sort the frequencies in decreasing order; the summation Σi f xi Wyi is terminated if a frequency f xi = 0 is encountered. This typically reduces the time spent in the summation, especially when sequences are closely related. As with Wyi, the sort order is computed once and cached. Observe that the roles of the two profiles are not symmetrical. It is most efficient to choose X, for which frequency sort orders are computed, to be the profile with the lowest amino acid diversity when averaged over columns. With this choice, the summation terminates earlier on average then if the other profile is identified as X. Note that out of N - 1 iterations of progressive alignment, a minimum of and maximum of N - 1 profile-profile alignments will include at least one profile containing one sequence only, and in the refinement phase exactly N of the 2N - 1 edges in the tree terminate on a leaf. At least half of all profile-profile alignments created in the MUSCLE algorithm therefore include a profile of one sequence only. Special cases where one or both profiles is a single sequence can be handled in separate subroutines, saving overhead due to unneeded loops that are guaranteed to execute once only. This optimization is especially useful for the LE function as it enables the logarithm to be incorporated into the W vector. Diagonal finding Many alignment algorithms are optimized for speed, typically at some expense in average accuracy, by using fast methods to identify regions of high similarity between two sequences, which appear as diagonals in the similarity matrix. The alignment path is then constrained to include these diagonals, reducing the area of the dynamic programming matrix that must be computed. MAFFT uses the fast Fourier transform to find diagonals. MUSCLE uses a different technique which we have previously shown [21] have comparable sensitivity and to be significantly faster. We use a compressed alphabet to find k-mers in common between two sequences, then attempt to extend the match. In the case of diagonal identification we found compressed alphabets to significantly out-perform the standard amino acid alphabet [21]. Currently, MUSCLE uses 6-mers in the Dayhoff alphabet for diagonal finding, as for the initial distance measure, though other alphabets are known to give slightly better performance [21]. A candidate diagonal is rejected if there is any overlap (i.e., if a single position in one of the sequences appears in two or more diagonals) or if it is less than a minimum length (default 24). The ends of the diagonal are deleted (by default, the first and last five positions) as they are less reliable. Despite these heuristics, we find the use of diagonal-finding to reduce average accuracy and to give only modest improvements in speed for typical input data; this option is therefore disabled by default. Similar results are seen in MAFFT; the most accurate MAFFT script is NWNSI [14], in which diagonal-finding is also disabled. Additive profiles Both the PSP and LE profile functions are defined in terms of amino acid frequencies and position-specific gap penalties. The data structure representing a profile is a vector of length LP in which each element contains frequencies for each amino acid type and a few additional values related to gaps. We call this data structure a profile vector, as distinct from a profile matrix, an explicit N × LP multiple alignment containing letters and indels. For N > 20, using profile vectors reduces the cost of computing the profile function compared with profile matrices, and is therefore preferred for use in dynamic programming. In CLUSTALW and MAFFT, the implementation of progressive alignment builds a profile matrix at each internal node of the tree, which is used to compute a profile vector. This procedure is O(NLP) = O(N2 + NL) in time and space, becoming expensive for large N. Observe that the count of a given amino acid in a column in the parent matrix is the sum of the counts in the two child columns that are aligned at that position (Figure 7). With a suitable sequence weighting scheme, it is therefore possible to compute the amino acid frequencies of the parent profile vector from the frequencies in the two child profile vectors and the alignment path. This is an O(LP) procedure in both time and space, giving a significant advantage for N >> 20. Three issues must be addressed to fully implement this idea: the sequence weighting scheme, inclusion of occupancy factors and position-specific gap penalties, and construction of a profile matrix (i.e., the final multiple alignment) at the root node. Sequence weighting For the frequencies in the parent profile vector to be a linear combination of the child frequencies, the weight assigned to a sequence must be the same in the child and parent profiles. This requirement is not satisfied, for example, by the Henikoff or PSI-BLAST schemes, which compute weights based on a multiple alignment. We therefore choose the CLUSTALW scheme, which computes a fixed weight for each sequence from edge lengths in the tree. Gap representation To compute gap penalties, we need the frequencies fo of gap opens and fc of gap closes in each position. In the case of the LE profile function, we additionally require the gap frequency fG. These can be accommodated by storing fo, fc and fe in the profile vector, where fe is the frequency of gap-extensions in the column (meaning that indels are found in a given sequence in the column, the preceding column and in the following column; i.e., a gap-close is not counted as an extension). These three occupancy frequencies are sufficient for computing the profile function and the position-specific gap penalties b and t. Note that we can compute the frequency fG of indels, as needed for the occupancy factor in the profile function, as follows: fG = fo + fc + fe.     (22) Now consider the problem of computing the occupancy frequencies in the parent profile vector, given only the child occupancy frequencies and the trace-back path for the alignment. Consider first a diagonal edge in the path, i.e. an edge that does not open or extend a gap, following another diagonal edge. In this case, the occupancy frequencies are computed similarly to amino acid frequencies (as a sum in which a child frequency is weighted according to the total weight of the sequences in its profile). For horizontal or vertical edges, i.e. edges that open or extend gaps, the parent occupancy frequencies can be computed by considering the effect of the new column of indels (Figure 8). It is straightforward to work through all cases and show that the three frequencies fo, fc and fe are sufficient for their values in the parent profile vector to be computed in O(LP) time from the child profile vectors and alignment path. Construction of the root alignment By avoiding the use of profile matrices, the complexity of a single progressive alignment iteration is reduced from O(LP 2 + NLp) space and O(LP2 + NLP) time to O(LP2) = O(L2 + NL) space and time. The NL term in the time complexity is now due only to the increase in profile length, and is therefore typically much smaller than before. The root alignment is constructed by storing the alignment path produced at each internal node. For each input sequence, the path to the root is followed, inserting the gaps induced by each alignment path at each internal node. This procedure is O(NLP log N) = O(N 2 log N + NL log N) time, and requires O(NLP) = O(NL + N 2) space for storage of the paths. This is expensive for large N, and we therefore optimize this step by using a device we call an e-string, a type of edit string. E-strings An alignment path can be considered as an operator on a pair of sequences that inserts indels into those sequences such that their lengths become equal. Conventionally, an alignment path is represented as a vector of three symbols representing edges in the graph, say M, D and I (for match, delete and insert, i.e. a diagonal, horizontal or vertical edge). Note that indels in one sequence are inserted only by Ds, indels in the other are inserted only by Is. Define an e-string e to be a vector of |e| integers interpreted as an operator that inserts indels into a string by scanning it from left to right (Figure 9). A positive integer n means skip n letters of the string; a negative integer -n means insert n indels at the current position. We require the vector to be in its shortest form, so signs always alternate. We represent an alignment path as a pair of e-strings, one for each sequence, assigned to the appropriate edges in the tree. We will typically find that |e|, the length of the e-string, is much less than LP, the length of the alignment path. Now consider the effect of applying two consecutive e-strings ("multiplying" them). This can be expressed as a third e-string, which can be efficiently computed in O(|e|) time from the multiplicands. For each leaf (input sequence), the product is computed of e-strings on the path to the root (Figure 10). The final e-string obtained at the root is then applied to the sequence. This method does not reduce the big-O time or space complexity, but is much faster than a naive implementation. Brenner's method Steven Brenner (personal communication) observed that a multiple alignment can be alternatively be obtained by aligning each sequence to the root profile. This requires O(NLP2) time, giving a lower asymptotic complexity in N at the expense of an additional factor of LP. This method gives opportunities for errors relative to the "exact" e-string solution (when a sequence misaligns to its copy in the profile), but can also lead to improvements by allowing the sequence to correctly align to conserved motifs that were not apparent when the sequence was added. (Note the resemblance to the refinement stage, which begins by re-aligning individual sequences to the rest). The chances for error are reduced by constraining the alignment to forbid gaps in the root profile. Our tests show that this method gives comparable average accuracy to the e-string solution but to be slower for up to at least a few thousand sequences, and e-strings are therefore used by default. Refinement complexity In the following, we assume that an explicit multiple alignment (profile matrix) of all sequences is maintained, and determine the complexity of each step in Stage 3. Step 3.1 determines the bipartition induced by deleting an edge from the tree. This is O(N) time, and sufficiently fast that there is little motivation for further optimization. Step 3.2 extracts profiles for the two partitions from the current multiple alignment and computes their profile vectors, which is O(NLP) time and space. Step 3.3 performs profile-profile alignment, which is O(LP2) time and space. Step 3.4 computes the SP score, which is O(N2LP) time and O(NLP) space (discussed in more detail shortly). A single iteration of Stage 3 is thus O(N2LP + LP2) time and O(NLP + LP2) space. There are O(N) edges in the tree, so executing this process for all edges is O(N3LP + NLP2) time and O(NLP + LP2) space, which is O(N4 + N3L + NL2) time and O(N2 + NL + L2) space. Assuming that a fixed maximum number of iterations of Stage 3 is imposed, this is also the total complexity of refinement. We now consider optimizations of the refinement stage. Anchor columns A multiple alignment can be divided vertically at high-confidence (anchor) columns. Each vertical block is then refined separately, improving speed and reducing space due to the O(L2) factor in dynamic programming. This strategy has been used by several previous algorithms, including PRRP [13], RASCAL [42] and MAFFT. In MUSCLE, the following criteria are used to identify anchor columns. The profile function (LE or PSP) must exceed a threshold, the averaged profile function over a window around the position must exceed a (lower) threshold, and the column may not contain a gap. In addition, the contribution to the averaged score from a single column has a ceiling, reducing skew in the averaged score due to exceptionally high-scoring columns. These heuristics are designed to avoid anchor columns that have high scores but are either artifacts (similar residues found by chance in unrelated regions) or are too close to variable regions. When performing a profile-profile alignment, each anchor column and its two immediate neighbors (which form the boundaries of vertical blocks) are required to remain aligned; i.e., terminal gaps are forbidden except at the true terminals. Introducing this constraint overcomes a small degradation in average alignment quality that is otherwise observed. This implies that the degradation is sometimes due to cases where a well-conserved region is divided into two parts by an anchor column, one of which becomes short enough that it misaligns to a similar short motif elsewhere. SP score Notice that computation of the SP score dominates the time complexity of refinement and of MUSCLE overall, introducing O(N4) and O(N3L) terms. We are therefore motivated to consider optimizations of this step. We first consider the contribution SPa to the SP score from amino acids; gap penalties require special treatment. Let s and t be sequences, x be a column, s [x] be the amino acid of sequence s in column x, and S(i, j) be the substitution score of amino acids i and j. It is convenient to impose an (arbitrary) ordering on the sequences and amino acid types. Then, SPa = Σx Σs Σt >s S(s [x], t [x]).     (23) Define δ(s, i, x) = 1 if s [x] = i, 0 otherwise, and ni [x] = Σs δ(s, i, x). We say ni [x] is the count of amino acid type i in column x. We can now transform the sum over pairs of sequences into a sum over pairs of amino acid types: SPa = Σx Σi ni Σj nj>i S(i, j) + 1/2 Σx Σi (ni2 - ni) S(i, i).     (24) Frequencies are computed as: f xi = ni [x]/ N.     (25) Using frequencies, For simplicity, we have neglected sequence weighting; it is straightforward to show that (26) applies unchanged if weighting is used. Note that (23) is O(N2LP) but (25) and (26) are O(NLP). For N >> 20, this is a substantial improvement. Let SPg be the contribution of gap penalties to SP, so SP = SPa + SPg. It is natural to seek an O(NLP) expression for SPg analogous to (26), but to the best of our knowledge no solution is known. Note that in MUSCLE refinement, the absolute value of the SP score is not needed; rather, it suffices to determine the difference in the SP scores before and after re-aligning a pair of profiles. Let SP(s, t) be the contribution to the SP score from a pair of sequences s and t, so SP = Σs Σt>s SP(s,t), and denote the two profiles by X and Y. Then we can decompose SP into intra-and inter-profile terms as follows: SP = Σs∈X Σt∈X:t>s SP(s, t) + Σs∈Y Σt∈Y:t>s SP(s, t) + Σs∈X Σt∈Y SP(s, t)     (27) Note that the intra-profile terms are unchanged in any alignment that preserves the columns of the profile intact, which is true by definition in profile-profile alignment. This follows by noting that any indels added to align the profiles are guaranteed to be external gaps with respect to any pair of sequences in the same profile. It therefore suffices to compute the change in the inter-profile term: SPXY = Σs∈X Σt∈Y SP(s, t).     (28) This reduces the average time by a factor of about two. We can further improve on this by noting that in the typical case, there are few or no changes to the alignment. This suggests computing the change in SP score by looking only at differences between the two alignments. Let π- be the alignment path before re-alignment and π+ the path after re-alignment. The change in alignment can be specified as the set of edges in π- or π+, but not both; i.e., by considering a path to be a set of edges and taking the set symmetric difference Δπ = (π- ∪ π+) - (π- ∩ π+). The path π+ after re-alignment is available from the dynamic programming traceback. The path π- before re-alignment can be efficiently computed in O(LP) time. Note that in order to construct the profile of a subset of sequences extracted from a multiple alignment, those columns that contain only indels in that subset must be deleted. The set of such columns in both profiles is therefore available as a side effect of profile construction, and this set immediately implies π-. It is a simple O(LP) procedure to compute Δπ from π- and π+. Note that SPa is a sum over columns, and there is a one-to-one correspondence between columns and edges in π. The change in SPa can therefore be computed as a sum over columns in Δπ, with a negative sign for edges from π-, reducing the time complexity from O(NLP) to O(N|Δπ|). We now turn our attention to SPg. We say that a gap G intersects Δπ if and only if any indel in G is in a column in Δπ, and denote by Γ the set of gaps that intersect Δπ. If a gap does not intersect Δπ, i.e. does not have an indel in a changed column, its contribution to SPg is unchanged. It therefore suffices to consider penalties for gaps in Γ, again with negative signs for edges from π-. The construction of Γ is straightforward in O(NLP) time. Finally, a sum over pairs in Γ is needed, reducing the O(N2) component to the smallest possible set of terms. Dimer approximation We next describe an approximation to SP that can be computed in O(NLP) time. Define a two-symbol alphabet {X, -} in which X represents any amino acid and - is the indel symbol. There are four dimers in this alphabet: XX, X-, -X and --, which denote by no-gap, gap-open, gap-close and gap-extend respectively. Re-write a multiple alignment in terms of these dimers, adopting the convention that dimer ab composed of symbol a in column x-1 and symbol b in column x is written in column x. Now consider the contribution to SPg of an aligned pair of dimers, written as ab↔cd. Clearly XX↔X- adds a gap-open penalty; XX↔-X adds a gap-close (Figure 11). To avoid double-counting, we will include only the penalty contribution of indels in the second column. Then XX↔X- adds a per-gap penalty, but XX↔-X adds zero because the second column does not contain a gap. External indels must be discarded; so, for example, --↔-- adds zero. In fact, aligning two identical dimers always contributes zero because any indel in the second column is found in both sequences and is therefore external. The contribution of all possible pairs of dimers is unambiguous, with the exception of -X↔--, which can add a per-gap or extend penalty (Figure 12). We approximate this case by assigning it a penalty of tg, where g is the default per-gap penalty and t is a tunable parameter, set to 0.2 by default. With this approximation, dimers can be treated like amino acids: the scores for each aligned pair of dimers forms a substitution matrix (Figure 13), and SPg can be computed by summing substitution scores over all pairs of sequences. We can now apply Equation 26, re-interpreting the frequency vectors f as having 24 components (20 amino acids and four dimers), and compute the change in SP by considering only those columns in Δπ. We find use of the dimer approximation to marginally reduce benchmark scores. By default, MUSCLE therefore uses the exact SP score for N ≤ 100 and the dimer approximation for N > 100, where the higher time complexity of the exact score becomes more noticeable. Evaluation of profile functions We have previously attempted a systematic comparison of profile functions [30]. The methodology used in that work demanded careful optimization of affine gap parameters for each function. This proved to be time-consuming and tedious, and we therefore tried the following alternative approach, inspired by the notion that a good profile function should be good at discriminating correctly aligned pairs of profile positions from incorrectly aligned pairs. The protocol begins with a set of pair-wise structural alignments. With the sequence of each structure as a query, we used PSI-BLAST to search the NCBI non-redundant protein sequence database [43], giving a multiple sequence alignment (profile) for each structure. Note that we use the term profile in this context to mean the sequence alignment produced by PSI-BLAST, not the scoring matrix. Using the structural alignments as a guide, we then created a database in which columns from the PSI-BLAST profiles were aligned to each other, giving a large set of pairs of alignment columns that we consider to be correctly aligned (the "true" database, although there are undoubtedly misaligned sequences and hence some incorrect pairs). By selecting the same number of pairs of columns at random from structures in different FSSP families, we created a similar ("false") database of unrelated pairs. A profile function was evaluated by computing the score of each pair of columns in both the true and false databases, and then sorting the results in order of increasing score. The results can be displayed by reviewing the sorted list and, for each score S in the list, plotting the number of true pairs with score ≤ S (x axis) against the number of false pairs with score ≤ S (y axis); we call the resulting graph a discrimination plot. Ideally, all true pairs would score higher than all false pairs, in which case the profile function would be a perfect discriminator and would always produce perfect alignments. A function that perfectly discriminates will appear as a Γ-shaped plot; a function that has no ability to discriminate will appear as a diagonal plot along the line x = y. If a function F has a discrimination plot that is always above another function G (i.e., DF(x) > DG(x) ∀ x, where DF is the discriminator plot for F as a function of x), then F has a superior ability to discriminate true from false pairs compared with G. If the plots intersect, the situation is ambiguous and neither function is clearly superior. We used sets of structural alignments from [30] (PP) and [44] (PP2). PP contains 588 structure pairs with sequence identity ≤ 30%, z-score ≥ 15, RMSD ≤ 2.5Å and an alignment length of ≥ 50 positions. These criteria were designed to select pairs of structures with low sequence identity and high structural similarity. PP2 contains 500 pairs selected from the FSSP database [45] with ≤ 30% sequence identity, z-score ≥ 8 and ≤ 12, RMSD ≤ 3.5Å and alignment length ≥ 50. The criteria for PP2 were designed to select challenging alignments with low sequence identity and relatively high structural divergence, leading to a high frequency of gaps and therefore, presumably, a stronger dependence on accurate identification of sequence similarity. Results on PP2 show the LE function to have higher discrimination than all other tested functions (historically, the LE function was designed by systematic trial and error using a wide range of different profile functions with feedback from discrimination plots). This is illustrated in Figure 14, in which the discrimination plot for LE on PP2 is compared with several other functions: PSP, LA, Yona and Levitt's [46], LAMA [47]. Using PP, we again find that LE is superior to LA (not shown), but the comparison with PSP is ambiguous as the discrimination plots intersect (Figure 15). A major advantage of this approach is that no gap penalties are required, with the result that once the databases have been constructed, a new function can be tested in seconds rather than the days or weeks that were needed with the earlier methodology. However, some caveats are in order. We are using PSI-BLAST as a gold standard for creating profiles, but PSI-BLAST may introduce biases both due to its selection of sequences for inclusion in the profile and due to errors in alignments of those sequences to the query. If the profile function will be used to align PSI-BLAST profiles, then this is an appropriate experimental design. But in the case of multiple sequence alignment, where profiles are produced iteratively by the profile function itself, the results may not be directly applicable. We also note that any monotonic transformation of the profile function leaves the discriminator plot unchanged as it does not change the sort order of the scores. (A monotonic transformation is F' = m(F) where m(x) is a monotonically increasing function). However, a monotonic transformation may change the alignments produced by a profile function, so we can regard high discrimination as a necessary but not sufficient condition for a good profile function. One can turn this into a virtue by noting that the discrimination plot allows the relative probability of true versus false to be determined from a score. It is therefore possible to numerically determine a log-odds function from the discrimination plot, which can be evaluated by table look-up. Using discrimination plots for PP2, we found the optimal transformation for LE to be close to linear, in contrast to other functions we tried, including PSP (results not shown). This observation further encouraged us to explore the performance of LE in an MSA algorithm. Testing on multiple alignment benchmarks we find LE to give superior results on BAliBASE, but statistically indistinguishable results on other databases (results not shown). MUSCLE therefore uses LE as the default choice as it sometimes gives better results but has not been observed to give lower average accuracy on any of our tests. It is also useful to introduce a method with a distinctively different scoring scheme as an alternative that may give better results on some input data and may provide unique features for incorporation into jury or consensus systems. One drawback of LE is its relatively slow performance due to the need to compute a logarithm for each cell of the dynamic programming matrix. Complexity of MUSCLE The complexity of MUSCLE is summarized in Table 2. We assume LP = O(L + N), the e-string construction for the root alignment, and a fixed number of refinement iterations. Results MUSCLE offers a variety of options that offer different trade-offs between speed and accuracy. In the following, we report speed and accuracy results for three sets of options: (1) the full MUSCLE algorithm including Stages 1, 2 and 3 with default options; (2) Stages 1 and 2 only, using default options (MUSCLE-prog); and (3) Stage 1 only using the fastest possible options (MUSCLE-fast), which are as follows: FBinary is used as a distance measure (Equation 2), the PSP profile function is used, and diagonal finding is enabled. Alignment accuracy In Tables 3 and 4 we report the speed and accuracy of MUSCLE v3.3, CLUSTALW v1.82, Progressive POA, a recently published method that is claimed to be 10 to 30 times faster than CLUSTALW for large alignments [48], and the MAFFT script FFTNS1 v3.82, the fastest previously published method known to us. On the advice of one of the authors of Progressive POA, we used command-line options selecting global alignment with truncated gap scoring (Catherine Grasso, personal communication). We report results both using distance matrices computed by BLAST (POA-blast) and using the distance method built into the program (POA). We use four sets of reference alignments: BAliBASE v2, PREFAB v3, SABmark v1.61, and a version of SMART from July 2000. The accuracy score is Q, the number of residue pairs correctly aligned divided by the length of the reference alignment. For more discussion of the reference data, assessment methodology and a comparison of MUSCLE with T-Coffee and NWNSI, the most accurate MAFFT script, see [2]. Execution speed To compare speeds for a larger number of sequences, we created a test set by using PSI-BLAST to search the NCBI non-redundant protein sequence database for hits to dienoyl-coa isomerase (1dci in the Protein Data Bank [49]), selecting the highest-scoring 1,000 sequences. This set of sequences had average length 282, maximum length 454 and average pair-wise identity 20%. We aligned randomly chosen subsets of from 200 to 1,000 sequences with each program and noted the total execution time. In the case of 1,000 sequences, the resulting alignments had from 1,100 from 1,400 columns, confirming that it is unrealistic to assume that LP is O(L). Results are shown in Figure 16. We have previously shown that MUSCLE-prog is faster than FFTNS1 on a set of 5,000 sequences, for which we estimated that CLUSTALW would require approximately one year [2]. In this test, MUSCLE-fast is approximately 3× faster than FFTNS1 for 200 sequences, and 5× faster for 1,000 sequences. This trend continues for larger numbers of sequences (complete results not shown), showing that MUSCLE-fast has lower asymptotic complexity, due largely to the use of additive profiles for progressive alignment compared with the profile matrices constructed by FFTNS1. Conclusions MUSCLE demonstrates improvements in accuracy and reductions in computational complexity by exploiting a range of existing and new algorithmic techniques. While the design–typically for practical multiple sequence alignment tools–arguably lacks elegance and theoretical coherence, useful improvements were achieved through a number of factors. Most important of these were selection of heuristics, close attention to details of the implementation, and careful evaluation of the impact of different elements of the algorithm on speed and accuracy. MUSCLE enables high-throughput applications to achieve average accuracy comparable to the most accurate tools previously available, which we expect to be increasingly important in view of the continuing rapid growth in sequence data. Availability and requirements MUSCLE is a command-line program written in a conservative subset of C++. At the time of writing, MUSCLE has been successfully ported to 32-bit Windows, 32-bit Intel architecture Linux, Solaris, Macintosh OSX and the 64-bit HP Alpha Tru64 platform. MUSCLE is donated to the public domain. Source code and executable files are freely available at . Figures and Tables Figure 1 Progressive alignment. Sequences are assigned to the leaves of a binary tree. At each internal (i.e., non-leaf) node, the two child profiles are aligned using profile-profile alignment (see Figure 2). Indels introduced at each node are indicated by shaded background. Figure 2 Profile-profile alignment. Two profiles (multiple sequence alignments) X and Y are aligned to each other such that columns from X and Y are preserved in the result. Columns of indels (gray background) are inserted as needed in order to align the columns to each other. The score for aligning a pair of columns is determined by the profile function, which should assign a high score to pairs of columns containing similar amino acids. Figure 3 Gap penalties in the SP score This figure shows a multiple alignment of three sequences s, t and u. The SP score is the sum over all pairs of sequences of their pairwise alignment score. The contribution to the SP score from the pair (s, t) is computed by discarding columns in which both sequences have indels (arrows). Such indels are said to be external with respect to the pair. Gaps in the remaining columns (gray background) are assessed affine penalties g + λe where g is the per-gap penalty, λ is the gap length, and e is the gap extension penalty. Figure 4 Position-specific gap penalties. An alignment of two profiles X and Y. Gaps in sequences t and u are embedded in X. Y contains a single sequence w. The gap in w (gray background) is inserted to align the profiles and is not part of Y. Consider the SP score for this alignment. We need not consider pairs of sequences in X as their scores are unchanged under all possible alignments of X to Y, leaving the inter-profile pairs (s, w), (t, w), (u, w) and (v, w). Note that there is no gap penalty for the pairs (u, w) and (v, w) as these pairs do not have gaps relative to each other. The remaining pairs (t, w) and (u, w) are assessed a penalty g + 3e for the gap in Y. The total over all pairs of open or close penalties due to a gap in Y is thus reduced in proportion to the fraction of sequences in X having a gap with the same open or close position. We incorporate this into the PSP score by using position-specific gap penalties b(x) and t(x). For example, b(x) in column 4 of X is half the default value because half of the sequences in X open a gap in that column. Note that there is no open penalty at the N-terminal and no close penalty at the C-terminal. This causes terminal gaps to receive half the penalty of internal gaps. Figure 5 Tree comparison. Two trees are compared in order to identify those nodes that have the same branching orders within subtree rotation (white). If a progressive alignment has been created using to the old tree, then alignments at these nodes can be retained as the same result would be produced at those nodes by the new tree. New alignments are needed at the changed (black) nodes only. Figure 6 Neighbor-joining and UPGMA trees for progressive alignment. Here we show the same set of four sequences and the order in which they will be aligned according to a neighbor-joining tree (above) and a UPGMA tree (below). Notice that t and u are the most closely related pair, but (s, t) and (u, v) are evolutionary neighbors. With neighbor joining, t and u are not aligned to each other until the root, in contrast to UPGMA, which aligns s and t as the first pair. Figure 7 Additive profiles. The profile functions in MUSCLE require amino acid frequencies for each column. Here we show the alignment of two profiles X and Y, giving a new profile Z. Note that the count nZi for amino acid i in a given column of Z is the sum of the counts in the child profiles, i.e. nZi = nXi + nYi. In terms of frequencies, this becomes f Zi = NXf Xi /NZ + NYf Yi/NZ, where NX, NY, NZ are the number of sequences in X, Y and Z respectively. Therefore, given a suitable sequence weighting scheme, it is possible to compute frequencies in Z from the frequencies in X and Y. This avoids the step of building an explicit multiple alignment for Z in order to compute frequencies, as done in CLUSTALW and MAFFT. Figure 8 Occupancy frequencies in additive profiles. Here we show an alignment of profiles X and Y giving Z. A column C of indels (shaded background) has been inserted at position x in order to align X to Y. To compute the number of gap-extensions in column x of Z, three cases must be considered: (1) a gap-extension in the corresponding column of Y, (2) a gap-open in the preceding column of X, and (3) a gap-extension in the preceding column of X. By enumerating all such cases, it is straightforward to compute the occupancy frequencies in Z from the occupancy frequencies of X and Y, plus the alignment path. Figure 9 E-strings. (1) The effect of the e-string operator <3,-1,2> on the sequence MQTIF. A positive number n skips n letters, a negative number -n insert n indels. (2) The effect of applying two successive e-strings. In the last line, the result is expressed as a new e-string applied to the original string. (3) We define multiplication on two e-strings as yielding the e-string that is equivalent to applying the two e-strings in order. (4) An alignment path is conventionally represented as a vector of edge types (M, D and I). In this example, MDMIMM, shown above a pairwise alignment, is the path that generates that alignment. The alignment can also be generated by a pair of e-strings (shown to the right). An alignment path is therefore equivalent to a pair of e-strings. Figure 10 Root alignment construction. Here we show the same progressive alignment as Figure 1. Each edge in the tree is labeled with the e-string for its side of the alignment at the parent node. The e-string needed to insert indels into a sequence in the root alignment can be determined by multiplying e-strings along the path to the root. For example, for sequence LSF, the root e-string is <3,-1,1>*<1,-1,2> = <1,-1,1,-1,1>. Figure 11 Dimers in the {X,-} alphabet. Gap penalties for the sequence pair (s, u) can be computed be considering all aligned pairs of dimers in the alphabet {X,-}, where X is any amino acid and - is the usual indel symbol. Four cases are highlighted. Note that an aligned pair of identical dimers never contribute a gap penalty as any indels in the dimers are necessarily external, as in the left-most example. Figure 12 Problem dimer pair. The aligned dimer pair -X ↔ -- causes a problem because its gap penalty contribution cannot be computed without additional information. Note that the first column of indels is external; after this column is discarded, different penalties may be needed, as these two examples show. Figure 13 Dimer substitution matrix. This matrix specifies the contribution to the total gap penalty for a pair of sequences for each possible pair of aligned dimers. Here, g is the per-gap penalty, e is the gap-extension penalty. The problem case -X ↔ -- is approximated as tg, where t is a tunable parameter. Figure 14 Discrimination plot for PP2. The x axis is the number of true column pairs with scores ≤ S for some value S, as a fraction of the total number of true pairs; the y axis is the number of false column pairs with scores ≤ S, as a fraction of the total number of false pairs. The databases were constructed from the PP2 test set. Shown are discrimination plots for the log-expectation (LE), log-average (LA), Yona-Levitt (YL), LAMA, and profile sum of pairs (PSP) functions. The LE function shows higher discrimination over the entire range of scores than any other function we tested (complete results not shown). The poor performance of the "standard" PSP function is striking. PSP displays negative discrimination over some of its range where it falls below the diagonal (dashed line). Figure 15 Discrimination plot for PP. This is similar to Figure 13, except that the database was generated from the PP test set. Here we see an ambiguous result as the discrimination plots for LE and PSP intersect. Figure 16 Execution time as a function of N. This plot shows the execution time as a function of N (number of sequences) for the tested alignment methods. Input data is from 200 to 1,000 sequences in increments of 200. Average sequence length is 282, maximum length 454. Table 1 Complexity of CLUSTALW. Here we show the big-O asymptotic complexity of the elements of CLUSTALW as a function of L, the typical sequence length, and N, the number of sequences, retaining the highest-order terms in N with L fixed and vice versa. Step O(Space) O(Time) Distance matrix N2 + L N2L2 Neighbor joining N2 N4 Progressive (one iteration) NLP + LP = NL + L2 NLP + LP2 = N2 + L2 Progressive (total) NL + L2 N3 + NL2 TOTAL N2 + L2 N4 + L2 Table 2 Complexity of MUSCLE. Here we show the big-O asymptotic complexity of the elements of MUSCLE as a function of L, the typical sequence length, and N, the number of sequences, retaining the highest-order terms in N with L fixed and vice versa. Step O(Space) O(Time) K-mer distance matrix N2 + L N2L UPGMA N2 N2 Progressive (one iteration) LP2 = NL + L2 LP2 = N2 + L2 Progressive (root alignment) NLP = N2 + NL NLP log N = N2 log N + NL log N Progressive (N iterations + root) N2 + NL + L2 N3 + NL2 Refinement (one edge) NLP + LP2 = N2 + L2 N2LP + LP2 = N3+ L2 Refinement (N edges) N2 + L2 N4+ NL2 TOTAL N2 + L2 N4 + NL2 Table 3 Accuracy scores. The average accuracy, measured by the Q score, is reported for each method on each set of reference alignments. Method PREFAB BAliBASE SABmark SMART MUSCLE 0.648 0.896 0.430 0.856 MUSCLE-prog 0.634 0.883 0.427 0.837 FFTNS1 0.619 0.844 0.376 0.815 MUSCLE-fast 0.616 0.849 0.396 0.813 CLUSTALW 0.588 0.860 0.404 0.823 POA-blast 0.577 0.839 0.352 0.788 POA 0.576 0.834 0.280 0.797 Table 4 CPU times. The total CPU time required to create all alignments in each test set, measured in seconds on a 2.5 GHz Pentium 4 desktop computer. Method PREFAB BAliBASE SABmark SMART MUSCLE-fast 540 11 45 30 FFTNS1 720 16 70 46 MUSCLE-prog 3,000 52 429 180 MUSCLE 11,000 81 1,500 560 POA-blast 11,000 90 290 670 CLUSTALW 15,000 160 210 480 POA 24,000 130 380 880 ==== Refs Notredame C Recent progress in multiple sequence alignment: a survey Pharmacogenomics 2002 3 131 144 11966409 Edgar RC MUSCLE: multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res 2004 32 1792 1797 15034147 10.1093/nar/gkh340 Wang L Jiang T On the complexity of multiple sequence alignment J Comput Biol 1994 1 337 348 8790475 Waterman MS Smith TF Beyer WA Some biological sequence metrics Adv in Math 1976 20 367 387 Lawrence CE Altschul SF Boguski MS Liu JS Neuwald AF Wootton JC Detecting subtle sequence signals: a Gibbs sampling strategy for multiple alignment Science 1993 262 208 214 8211139 Hogeweg P Hesper B The alignment of sets of sequences and the construction of phyletic trees: an integrated method J Mol Evol 1984 20 175 186 6433036 Feng DF Doolittle RF Progressive sequence alignment as a prerequisite to correct phylogenetic trees J Mol Evol 1987 25 351 360 3118049 Thompson JD Higgins DG Gibson TJ CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 1994 22 4673 4680 7984417 Notredame C Higgins DG Heringa J T-Coffee: A novel method for fast and accurate multiple sequence alignment J Mol Biol 2000 302 205 217 10964570 10.1006/jmbi.2000.4042 Bahr A Thompson JD Thierry JC Poch O BAliBASE (Benchmark Alignment dataBASE): enhancements for repeats, transmembrane sequences and circular permutations Nucleic Acids Res 2001 29 323 326 11125126 10.1093/nar/29.1.323 Thompson JD Plewniak F Poch O BAliBASE: a benchmark alignment database for the evaluation of multiple alignment programs Bioinformatics 1999 15 87 88 10068696 10.1093/bioinformatics/15.1.87 Hirosawa M Totoki Y Hoshida M Ishikawa M Comprehensive study on iterative algorithms of multiple sequence alignment Comput Appl Biosci 1995 11 13 18 7796270 Gotoh O Significant improvement in accuracy of multiple protein sequence alignments by iterative refinement as assessed by reference to structural alignments J Mol Biol 1996 264 823 838 8980688 10.1006/jmbi.1996.0679 Katoh K Misawa K Kuma K Miyata T MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform Nucleic Acids Res 2002 30 3059 3066 12136088 10.1093/nar/gkf436 Van Walle I Lasters I Wyns L Align-m–a new algorithm for multiple alignment of highly divergent sequences Bioinformatics 2004 Schultz J Copley RR Doerks T Ponting CP Bork P SMART: a web-based tool for the study of genetically mobile domains Nucleic Acids Res 2000 28 231 234 10592234 10.1093/nar/28.1.231 Ponting CP Schultz J Milpetz F Bork P SMART: identification and annotation of domains from signalling and extracellular protein sequences Nucleic Acids Res 1999 27 229 232 9847187 10.1093/nar/27.1.229 Schultz J Milpetz F Bork P Ponting CP SMART, a simple modular architecture research tool: identification of signaling domains Proc Natl Acad Sci U S A 1998 95 5857 5864 9600884 10.1073/pnas.95.11.5857 Vinga S Almeida J Alignment-free sequence comparison-a review Bioinformatics 2003 19 513 523 12611807 10.1093/bioinformatics/btg005 Dayhoff MO Schwartz RM Orcutt BC Dayhoff MO, Ech RV A model of evolutionary change in proteins in Atlas of protein sequence and structure, 1978 Maryland: National Biomedical Research Foundation Edgar RC Local homology recognition and distance measures in linear time using compressed amino acid alphabets Nucleic Acids Res 2004 32 380 385 14729922 10.1093/nar/gkh180 Kimura M The neutral theory of molecular evolution 1983 Cambridge University Press Saitou N Nei M The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 1987 4 406 425 3447015 Sneath PHA Sokal RR Numerical taxonomy 1973 San Francisco: Freeman Thompson JD Higgins DG Gibson TJ Improved sensitivity of profile searches through the use of sequence weights and gap excision Comput Appl Biosci 1994 10 19 29 8193951 Henikoff S Henikoff JG Position-based sequence weights J Mol Biol 1994 243 574 578 7966282 10.1016/0022-2836(94)90032-9 Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W Lipman DJ Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Nucleic Acids Res 1997 25 3389 3402 9254694 10.1093/nar/25.17.3389 Gerstein M Sonnhammer EL Chothia C Volume changes in protein evolution J Mol Biol 1994 236 1067 1078 8120887 10.1016/0022-2836(94)90012-4 Gotoh O A weighting system and algorithm for aligning many phylogenetically related sequences Comput Appl Biosci 1995 11 543 551 8590178 Edgar RC Sjolander K A comparison of scoring functions for protein sequence profile alignment Bioinformatics 2004 Sjolander K Karplus K Brown M Hughey R Krogh A Mian IS Haussler D Dirichlet mixtures: a method for improved detection of weak but significant protein sequence homology Comput Appl Biosci 1996 12 327 345 8902360 Altschul SF Amino acid substitution matrices from an information theoretic perspective J Mol Biol 1991 219 555 565 2051488 Jones DT Taylor WR Thornton JM The rapid generation of mutation data matrices from protein sequences Comput Appl Biosci 1992 8 275 282 1633570 Muller T Spang R Vingron M Estimating amino acid substitution models: a comparison of Dayhoff's estimator, the resolvent approach and a maximum likelihood method Mol Biol Evol 2002 19 8 13 11752185 von Ohsen N Zimmer R Gascuel O, Moret BME Improving profile-profile alignment via log average scoring Algorithms in Bioinformatics, First International Workshop, WABI 2001 2001 2149 Berlin: Springer-Verlag 11 26 Thompson JD Plewniak F Poch O A comprehensive comparison of multiple sequence alignment programs Nucleic Acids Res 1999 27 2682 2690 10373585 10.1093/nar/27.13.2682 Myers EW Miller W Optimal alignments in linear space Comput Appl Biosci 1988 4 11 17 3382986 Felsenstein J Inferring Phylogenies 2004 Sunderland, Massachusetts: Sinauer Associates Sauder JM Arthur JW Dunbrack RL Jr Large-scale comparison of protein sequence alignment algorithms with structure alignments Proteins 2000 40 6 22 10813826 10.1002/(SICI)1097-0134(20000701)40:1<6::AID-PROT30>3.0.CO;2-7 Durbin R Eddy S Krogh A Mitchison G Biological sequence analysis 1998 Cambridge University Press Setubal J Meidanis J Introduction to computational biology 1997 Pacific Grove, California: Brooks/Cole Thompson JD Thierry JC Poch O RASCAL: rapid scanning and correction of multiple sequence alignments Bioinformatics 2003 19 1155 1161 12801878 10.1093/bioinformatics/btg133 Pruitt KD Tatusova T Maglott DR NCBI Reference Sequence project: update and current status Nucleic Acids Res 2003 31 34 37 12519942 10.1093/nar/gkg111 Edgar RC Sjolander K COACH: profile-profile alignment of protein families using hidden Markov models Bioinformatics 2004 Holm L Sander C Touring protein fold space with Dali/FSSP Nucleic Acids Res 1998 26 316 319 9399863 10.1093/nar/26.1.316 Yona G Levitt M Within the twilight zone: a sensitive profile-profile comparison tool based on information theory J Mol Biol 2002 315 1257 1275 11827492 10.1006/jmbi.2001.5293 Pietrokovski S Searching databases of conserved sequence regions by aligning protein multiple-alignments Nucleic Acids Res 1996 24 3836 3845 8871566 10.1093/nar/24.19.3836 Grasso C Lee C Combining partial order alignment and progressive multiple sequence alignment increases alignment speed and scalability to very large alignment problems Bioinformatics 2004 Berman HM Westbrook J Feng Z Gilliland G Bhat TN Weissig H Shindyalov IN Bourne PE The Protein Data Bank Nucleic Acids Res 2000 28 235 242 10592235 10.1093/nar/28.1.235

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1191533934510.1186/1471-2105-5-119Methodology ArticleBayesian model accounting for within-class biological variability in Serial Analysis of Gene Expression (SAGE) Vêncio Ricardo ZN [email protected] Helena [email protected]ão Diogo FC [email protected] Carlos AB [email protected] Statistics Department, Instituto de Matemática e Estatística – Universidade de São Paulo, Rua do Matão 1010, 05508-090 São Paulo, BRAZIL2 BIOINFO-USP – Núcleo de Pesquisas em Bioinformática da Universidade de São Paulo, Rua do Matão 1010, 05508-090 São Paulo, BRAZIL3 Ludwig Institute for Cancer Research – São Paulo Branch, Rua Prof. Antônio Prudente 109, 01519-010 São Paulo, BRAZIL4 Hospital do Câncer A.C. Camargo, Rua Prof. Antônio Prudente 109, 01519-010 São Paulo, BRAZIL2004 31 8 2004 5 119 119 14 5 2004 31 8 2004 Copyright © 2004 Vêncio et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background An important challenge for transcript counting methods such as Serial Analysis of Gene Expression (SAGE), "Digital Northern" or Massively Parallel Signature Sequencing (MPSS), is to carry out statistical analyses that account for the within-class variability, i.e., variability due to the intrinsic biological differences among sampled individuals of the same class, and not only variability due to technical sampling error. Results We introduce a Bayesian model that accounts for the within-class variability by means of mixture distribution. We show that the previously available approaches of aggregation in pools ("pseudo-libraries") and the Beta-Binomial model, are particular cases of the mixture model. We illustrate our method with a brain tumor vs. normal comparison using SAGE data from public databases. We show examples of tags regarded as differentially expressed with high significance if the within-class variability is ignored, but clearly not so significant if one accounts for it. Conclusion Using available information about biological replicates, one can transform a list of candidate transcripts showing differential expression to a more reliable one. Our method is freely available, under GPL/GNU copyleft, through a user friendly web-based on-line tool or as R language scripts at supplemental web-site. ==== Body Background An important challenge in Serial Analysis of Gene Expression (SAGE) [1] analysis is the decision whether a gene is differentially expressed between two classes, for example tumoral vs. normal classes. In statistical terms, this essential step is to test the null hypothesis H0: "gene has no differential expression between the two probed classes". A much more usual approach is to assign an index (P-value or Bayes factor, for example) that measures the confidence/significance of the hypothesis and let the biologists themselves to establish a cutoff of what they call significant. This necessity arises because counting sequenced SAGE tags is a process prone to random and systematic errors that affect gene expression abundance estimates. Systematic errors may come from various sources such as GC content bias [2], sequencing errors [3,4] as well as the possibility of non unique tags. This kind of error can be detected/corrected using some bioinformatics procedures such as quality control of automatic sequencing pipe-line [5], or statistical estimation procedures such as "denoising" [6,7]. Random errors are due to the inherent stochastic characteristic of SAGE data acquisition: sampling from automatic sequencing. Like colored balls in an urn, sampling and counting SAGE tags from a library is commonly modeled by a Bernoulli Process relying on an infinite population sampling approximation. If an Expressed Sequence Tag (EST) library is non-normalized, its counting data, also known as "Digital-Northern", reflects the abundance of genes. Likewise, the Massively Parallel Signature Sequencing (MPSS) [8] technique counts tags to infer the transcriptome, but using a completely different strategy from traditional DNA sequencing methods, that allows augmented high-throughput capability. Therefore, all the results discussed here are readily applicable to "Digital-Northern" or MPSS context since, from a mathematical viewpoint, all represent the same bioinformatics problem: counting transcripts (as balls in urns). Nowadays, the variability in SAGE abundance data is modeled only as due to sampling from sequencing, since almost all statistical procedures are performed after aggregation of observations from various libraries of the same class, creating a "pseudo-library". See [9-11] for good reviews on statistical techniques used in SAGE analysis. This extensively used trick tacitly ignores the within-class variability, i.e., the biological variability among individuals within a class (different patients having the same cancer diagnosis, for example), and could lead to overconfident conclusions. Results Here we propose a Bayesian model of mixtures to account for within-class variability as a generalization of the Beta-Binomial model [12]. We also show that the usual "pseudo-library" construction is a particular case of our mixture model. Finally, we propose the use of the Bayes Error Rate to intuitively rank the differential expression hypothesis under a Bayesian framework, avoiding several technicalities and difficulties such as: typeI and typeII error analysis, Bonferroni-like multiple testing correction, asymptotic results evocation, imposition of a test statistic and null probability density function (pdf), and so on. Statistical model The counting process from automatic sequencing of one single i-th library is often modeled as a Bernoulli Process and a fixed unknown tag abundance πi is implicitly assumed. The pdf of the random variable of interest, "expression abundance" π ∈ [0;1] among all n libraries is unknown, thus each library could be regarded as being created by a realization of π. These features lead naturally to mixture models [13,14]: where: f(·) is the unknown pdf of the abundance among same-class libraries parameterized by a vector θ, X = (x1,..., xn) is the vector of counts in all n libraries of same class, M = (m1,..., mn) is the vector of library sizes and L is the likelihood of each i-th observation. The common procedure of merging all observations from libraries of the same class, constructing a "pseudo-library" before statistical inference, is recognized as a particular case of this mixture model: just assume that all libraries have strictly the same abundance, with no biological variability. Mathematically, this is a function with infinite probability density over one single abundance value π = θ and zero over every other π ≠ θ, or a Dirac's Delta function. Using f(·) as a Dirac's Delta function constrained to [0;1], turn Eq.1 into the familiar and commonly used binomial distribution (see derivation in the Methods section). We believe that Dirac's Delta is a naive description of real-life SAGE libraries. The Beta distribution is an alternative with non-zero within-class variance to account for intuitively expected biological differences among them. Using f(·) as a Beta in Eq.1, yields the so-called Beta-Binomial model (see derivation in the Methods section). Given the parameter vector θ that describes the random variable π of some fixed gene G, we must decide if there is a difference between A and B classes (e.g, tumor vs. normal classes). We propose to consider genes as being differentially expressed based on non-superposition of the predictive Beta pdfs of both A and B classes. By "predictive" we mean that we use the a posteriori mode in the Beta pdfs. The "non-superposition" intuitive feature is mathematically written as the Bayes Error Rate E [15]: where f(·) is the Beta pdf and "hat" over the parameters indicate the values that lead to an a posteriori pdf maximum. The a posteriori distribution is obtained as usual from Bayesian Statistical Theory (a priori pdf choice and detailed derivation are in the Methods section). Intuitively, if the pdfs are "far apart", the gene probably has reproducible differential expression between classes. In this case, rarely could one misclassify class A as B and vice-versa. Figure 1 gives some insight about this fact. Using our proposed approach, the "far apart" notion means a small Bayes Error Rate E. For adepts of the Frequentist Statistics, this evidence measure could resemble a typeI and typeII errors sum, however it is just an illustrative analogy. As in any significance test method, the experimenter must define what is a high significance E value. This cutoff should be guided by external and independent confirmatory assays. To avoid crude decision boundaries, one could rank their significance results but there is no way to avoid some arbitrariness in any kind of statistical test. In the classical Frequentist Statistics framework, it is common to call a result as significant if it presents a P-value ≤ 0.01 in a t-like-test, hoping that this could control the error at this level. However, due to technical difficulties such as lack of sensitivity of posterior confirmatory methods or high absolute expression (not differential expression) necessity, this apparent statistically sound results could be not useful in a pragmatic sense. That is why we prefer to rank the differential expression results and allow researchers to establish a cutoff compatible with their subsequent application for the selected genes, rather than split them based in assumption-derived error-rate cutoffs. People familiar with the Frequentist Statistics framework could miss multiple testing considerations, typeI/typeII error studies, and so on. However, in the Bayesian framework, several of these concerns are meaningless since we work with parameters space and not with sample space. The bayesians avoid statements about "data that could be observed but was not" and work only with available information (prior and experimental), extracting all possible information from data effectively observed. For those genes classified as differentially expressed, one should aggregate intuitive information adding "error-bars" to expression ratios. Recently we have developed a method to add credibility intervals to gene expression ratio [16], which could improve posterior analyses such as clustering [17] or comparison with microarray data. Comparison with available methods using publically available data To show the model is usefulness, we applied it to a tumor vs. normal two-classes comparison problem. We chose a subset of brain tumor SAGE data from The Cancer Genome Anatomy Project's SAGE Genie public database web-site [18]. The SAGE Genie performs several bioinformatics protocols to assure the quality of its data with systematic errors cleaning/correction [19]. We used all 4 available libraries in SAGE Genie until Jan/2004 from astrocytoma grade III tumors and almost all (except the fetal library) normal brain libraries (see Methods section for details about libraries). We want to stress 3 typical and important cases: (i) when our measure agreed with other evidence measures accepting null hypothesis H0, i.e., there is no evidence of differential behavior between tumor and normal classes; (ii) when our method agreed with others rejecting H0, i.e., there is evidence of differential expression; and (iii) when our method showed evidence in favor but other evidence measures showed evidence against the H0. Case (iii) is the main motivation of our method since it reveals situations that researchers may call a gene differentially expressed and, in fact, it could be not so significant if biological replicates are taken into account. The other evidence measures used were: the Audic-Claverie bayesian evidence [20], the classical Fisher Exact Test P-value, and the classical χ2 P-value, all obtained using the IDEG6 web-interface [21,22] (see Methods section). A case (i) prototype is the TTTCAATAGA tag with XT = (0, 2, 5, 8) and XN = (1, 1, 0, 0, 0, 7, 2). The Audic-Claverie, Fisher and χ2 methods yield P-values of 0.06, 0.44, 0.41, respectively, indicating low evidence against H0 for all mystical significance level cutoffs ≤ 0.01, ≤ 0.05 or ≤ 0.1. The Bayes Error Rate evidence is E = 0.61, an intuitively unacceptable superposition level between the normal and tumoral predictive Beta pdfs, showing that there is no separable behavior between classes. Figure 2a shows an obvious superposition between pdf and observations of this two classes. A case (ii) prototype is the AAAAGAAACT tag with XT = (7, 11, 18, 10) and XN = (7, 1, 2, 1, 2, 0, 3). All P-values are 0.00 (zero), significant at any cutoff level. Our evidence is E = 0.03, showing safely that this gene behaves differentially between normal brain and astrocytoma grade III patients. Figure 2b shows that two Betas are apart from each other and, even observing clear within-class variability, the expression is different. A case (iii) prototype is the TTGGAGATCT tag with XT = (7, 239, 244, 123) and XN = (54, 27, 33, 21, 40, 196, 28). All P-values are 0.00 (zero), indicating significant difference between classes. On the other hand, our evidence E = 0.73 indicates high superposition between tumor and normal classes. Figure 2c shows that within-class variability for tumor class is not negligible. It is obvious that individual libraries confound their results with normal brain libraries, and the Betas have a relatively high intersection. Using a common "pseudo-library" approach, one is lead to call this gene as a strong discriminator between classes. We believe that this is a suspect conclusion. There are several other obvious case (iii) examples, such as tag TACAGTATGT in Figure 2d, that received P-values < 0.01 from all other methods, and they are the main concern of our method since they may lead to waste of resources in clinical validation efforts of genes that, by SAGE itself, could be left behind in favor of other promising genes. All tag results are available as additional file and graphics for all tags are at the supplemental web-site [23]. One could think about a case (iv) when considering within-class variability leads one to H0 rejection, but considering "pseudo-libraries" leads to H0 acceptance. This seems to be inconsistent since one expects that, once H0 is accepted in a simplified model, it should also be accepted in the complete model. In fact, we do not observe such a situation, except by tags with P-values or Bayes Error Rate very close to arbitrarily defined cutoff values. We believe that these occurrences are just "edge effect" manifestations. Discussion In order to assure that we are dealing with a fundamental question in SAGE analysis, we show more insights analyzing the method's robustness using the same data but excluding "small" libraries. Also, we draw some parallels between our proposed method and the only available published solution for dealing with within-class variability, a t-test approximation [12]. We used our method with all available libraries but some of them are smaller than 50,000 tags (see Table 1). In the SAGE community, libraries smaller than this arbitrary limit are considered "small". Several researchers claim that these are non-representative and should be excluded from analysis. We observed several case (iii) tag examples which remain as case (iii) if we use libraries with size > 40,000 and > 50,000 (shown at the supplemental web-site only). Figure 3 shows a tag example analyzed in these tree setups and it is clear that inclusion of "small" libraries gave pretty much the same result, indicating robustness of our method against small class size variations and against "small" sized libraries. Moreover, these libraries are not always outliers from biological sampling but seem to be samples like any other. These results suggest that one can use the "small" libraries, jointly with non-"small" ones, because biological variability seems to be greater than binomial sampling variability. Obviously, we are not recommending to use only "small" libraries in SAGE analysis, but suggesting that our method is relatively robust. For low expression genes, the binomial sampling variability should become more relevant as the library size decreases. Also, the results obtained using two/three libraries could be very different from using just one. These proprieties could be tag dependent since some tags could be much noisier than others for biological reasons. Some "denoising" procedure could be used before application of our method [7]. Therefore, our findings should be carefully interpreted. To prove that the incoherence of using "pseudo-libraries" methods is not a prerogative of tags showing small fold-changes, we analyzed another three very illustrative examples: ATGGCAACAG, GGATGTGAAA, and GTATGGGCCC; which are case (iii) tags. These tags present high fold changes: 7.59, 8.15 and 25.80 fold-change respectively, augmented in pooled tumor libraries. Using the well-known Fisher Exact test, χ2 classical test and the Audic-Claverie's method, we get 0.00 (zero!) for all P-values of the no differential expression null hypothesis. Using the conceptually different Bayesian P-value implemented at SAGE Genie [24,25] we obtain 0.01, 0.00 and 0.00 respectively for posterior probabilities of fold-changes greater than 4-fold. Finally, using our own proposed measure, applied to the pool, we get E = 0.00 meaning no superposition between the two classes pdfs. All these results indicate strong significance in differential expression of these tags. However, if we consider within-class variability, the test proposed by Baggerly et al. [12] yields 0.08, 0.07 and 0.15 respectively for t-test P-values, and our method yields Bayes Error Rates of 0.38, 0.37, 0.43 respectively; indicating not so significant evidence in favor of the differentially expressed hypothesis. A closer look at the graphics of these tags induces one to believe that there is no reproducible differential expression because several observations of tumor and normal are superimposed (all graphics available at supplemental web-site [23]). Since we show clearly that methods that use "pseudo-library" aggregation could be incoherent in some cases, a natural question is how our proposed method performs compared to the only published solution that accounts for within-class variability, the Baggerly et al. [12]t-test approximation. Without knowing the true state of all tags, it is impossible to carry out a serious benchmark. Since the interpretation of evidence measures is very different, the performance could be subjected to an arbitrary cutoff selection for each method. Figure 4 shows a scatter-plot of evidence measures obtained for each of the two methods. It is clear from this graphic that there are many more tags considered as differentially expressed using our method than the t-test approximation, considering E ≤ 0.1 and P-value ≤ 0.01. There are also some tags selected by t-test and ignored by ours. It is impossible to know which method perform better without the true unknown status of those tags. Looking at individual libraries results, constructed as depicted in Figure 2 for example, could help in this analysis but this is a subjective procedure. It is important to bear in mind that a difficulty is hidden in the Beta modeling imposed in the very first beginning. If Beta is not a good model for an unknown biological behavior, then some apparent inconsistency could appear in both Baggerly et al. [12] and our approaches. However, our general mixture model allows another propositions. Other simplex constrained pdfs, different from Beta, exist but the tractability is much more difficult [26]. We believe that to build a fully non-parametric approach to this problem is a very hard issue, but should be considered as a future challenge. Conclusion Until now, almost all statistical methods for SAGE data analysis tacitly ignore the within-class variability. To our knowledge, the firsts to formally address this issue was Baggerly et al. [12] who introduced the Beta-Binomial model as the correct way to model the probability of counting tags instead of Binomial models. They also proposed a t-like statistics, outlined a possible hypothesis test using the classical Frequentist Statistics framework and evoked some asymptotic results for t pdf justification. In this work we presented the Bayesian alternative for this problem and defined a theoretical model that views Baggerly's Beta-Binomial approach or even the common Binomial approach as particular cases of mixture models. Other models are possible modifying the mixing distribution, such as Beta-Poisson [14], or using other simplex constrained pdf [26] to model expression abundance. At last, but not at least, we proposed a method for ranking differentially expressed genes between two classes using the Bayes Error Rate as an intuitive measure of separation between the classes pdfs, avoiding statistical test formalism and its conceptual/practical difficulties. We show that there are cases in which approaches that ignore within-class variability will lead to high significance in differences between tumor and normal classes, but looking carefully at individual observations jointly, one should not attribute such high significance to them since abundance probability density functions have considerable superposition. In conclusion, we recommend that within-class variability must be taken into account in any statistical analysis of SAGE data if replicates are available. We suggest that biological replication should be considered in planning new SAGE experiments. Methods General bayesian model To start generically, suppose that the probability density function (pdf) for the random variable of interest "expression abundance" π ∈ [0;1] of some gene G is indexed in a model family by means of a parameter vector θ. Therefore, following the usual Bayesian framework, the a posteriori pdf that describes the class is: where: X = (x1,..., xn) is the vector of counts in all n libraries of same class, M = (m1,..., mn) is the vector of total observations in all n libraries of same class, g(·) is the a priori pdf, and L is the likelihood of each i-th observation. Note that the product of all likelihood functions over all observations is the so-called Likelihood Function. The counting process from automatic sequencing is often modeled as a Binomial. Since the sample size and the stopping rule are not known in advance the model is not strictly Binomial. We do not need the combinatorial constant in the model, but we write it just because it is commonly used and will vanish in posteriori expression anyway. "Pseudo-Library" method as particular case Merging all observations from the same class libraries and constructing "pseudo-libraries", with the sum of their components, is the standard procedure to use replicates. Our general model is reduced to this (unrealistic) one if one uses f(·) as a Dirac's Delta in Eq.1: where: 1{·} is the indicator function. Using Eq.4 in Eq.3 yield: where: g(θ) = 1, the non-informative uniform a priori distribution. The expert recognizes that θ ~ Beta(1 + Σxi, 1 + Σmi - Σxi), and the sum of observations is the mathematical translation of "pseudo-libraries" construction. Beta-Binomial method as particular case The only published solution that allows non-zero within-class variance in SAGE analysis is the Beta-Binomial model [12]. Using f(·) as a Beta in Eq.1 we get the Beta-Binomial model as a particular case of general model: where: B(·) is the beta special function, and: Again, an expert recognizes θ = (θ1, θ2) as the mean and standard deviation (stdv) of a Beta random variable. We prefer this parameterization of Beta distributions instead of the common (α, β) one because: (i) it is much more intuitive to biologists to deal with mean and stdv than with abstract α and β, and (ii) as α, β > 0, the domain Θ = {(θ1, θ2): 0 ≤ θ1 ≤ 1, 0 ≤ θ22 <θ1 (1- θ1) ≤ 1/4} is bounded and much more amenable to perform the necessary numerical computations. Using Eq.6 in Eq.3 yield: where: g(θ1, θ2) is the priori pdf. A Priori distribution definition To complete a Bayesian model, it is necessary to choose the a priori pdf. We use an uniform distribution over Θ. On the other hand, we know in advance that variance of this model cannot be smaller than the variance eventually obtained if we do not consider within-class variability. Even if the within-class variability is very small, it cannot be estimated as being smaller than the simple sampling error because they are inseparable, and sampling error is the lower limit [12]. As an illustration, the same situation could occur if one takes several diameter measurements of a folded paper ball and a perfect sphere using a common ruler. In the first case, the intrinsic nature of the measured object dominates the measurement variability but, in the second case, we cannot know the diameter of the perfect sphere with better precision than our ruler can measure. This kind of knowledge is naturally incorporated in Bayesian statistics by means of a priori distributions. To match our desired features, it is sufficient to define an uniform over the Θ parameter space but constrained at a minimum stdv σ, obtained from the result of no within-class variance model: over domain Θ = {(θ1, θ2): 0 ≤ θ1 ≤ 1, 0 ≤ θ22 <θ1 (1 - θ1) ≤ 1/4}. Since we showed (Eq.5) that the no within-class variance model is θ ~ Beta(1 + Σxi, 1 + Σmi - Σxi), it is easy to obtain our lower stdv boundary from Beta variance: Therefore, using Eq.9 and Eq.10 in Eq.8, our posteriori is completely defined. Differential expression detection We detect a tag as differentially expressed using the Bayes Error Rate E [15] in both predictive Beta pdfs: where: Note that f(·) is the Beta pdf, as in Eq.6 development. The "hat" over θ = (θ1, θ2) indicates values that leads Eq.8 to maximum. As usual, the maximization, subject to constrain Θ defined previously, is made upon logarithm of posteriori's core since it gives the same estimates as the posteriori itself: Figure 5 shows an example of this process. See Results section to get an intuitive notion of this evidence measure. Implementation – numerical analysis The method was implemented as R language [27,28] scripts which are freely available under GPL/GNU copyleft license at supplemental web site [23]. At this web page there are details on how to run it locally. Our method is computer-intensive mainly because some numeric maximization and integration are needed. We used efficient R built-in routines to perform such numerical tasks. Remember that maximization needed in Eq.12 is constrained, thus we used simply auxiliary re-parameterization to obtain linear constrains and used the constrOptim R routine. For numerical integration we used the 1-dimensional gaussian quadrature integrate R built-in function. Although numerical integration of Eq.9 should be performed in all [0;1] support, the relevant contribution for this integral is concentrated in a much smaller region. Integrating over the formal limits will cause serious numerical errors, and to avoid this problem we approximate our integration region to an interval delimited by 0.005 and 0.995 quantile of each Beta pdf since the relevant density lie in there. The credibility intervals ("error-bars") for the expression ratio of interesting tags were obtained as described in our recent work [16]. We chose arbitrarily 68% credibility intervals. Implementation – Web based interface We have also developed an easy-to-use web-based service that performs all calculations at our server and provides password-protected results. Although desirable, for the sake of automatic web hyperlink with SAGE Genie database, it is not necessary to explicitly identify the tags analyzed but rather any (custom) i.d. string. This could increase privacy or make our web-interface useful for "Digital-Northern", MPSS or any mathematically related problem of mixtures from binomial sampling. Figure 6 shows snapshots of the interface. Publically available data The Table 1 list the SAGE Genie's library name, Gene Expression Omnibus (GEO) [29] accession code and size of all used libraries. For our aims, it is sufficient to focus the analysis at the tag level. Thus, we process the tag counts and let the identification of tag's best gene match as a posterior question that could be carefully done only to really interesting tags. We choose not to process tags whose counts appear only in libraries of one class. It is important to note that all libraries are from bulk material, without cell-lines, and came from patients with similar disease description. The normal libraries came from different normal regions of the brain. We think that this data set is very illustrative since there are biological replicates in the tumor class allowing clear verification of within-class biological variability. On the other hand, taking only one kind of disease, astrocytoma grade III, instead of all brain tumors in the database, leads one to believe that the within-class variability is in fact due to biological diversity of the patients and not due to very distinct molecular profile of distinct brain tumors stored in SAGE Genie's database. Therefore, we believe that this in silico comparison is well-suited to demonstrate the necessity of dealing with within-class effect, although it is not our aim here to make a detailed or biological analysis of brain tumor data. Comparison with other methods To bring some intuition about our differential expression evidence measure, we tabulated evidence measure obtained from the famous Fisher Exact Test, the classical Pearson's χ2 proportion test and the bayesian Audic-Claverie's method. All these tests were performed using the easy-to-use web-interface IDEG6 [21,22]. The "P-values" are conceptually very different from our evidence measure but are the most used evidence measures. Although numbers cannot be compared, the conclusions obtained from these methods should be since graphical representation of each library observation gives clear indication of incoherence of "pseudo-library" methods. The results of the significance measures for all tags are available as additional Excel© or OpenOffice© interactive files in which the user can set cutoffs for the significance measures, and explore the differences in conclusions. We carry out a qualitative comparison of our method with Baggerly et al. [12]t-test approximation in a graphical way since it is impossible to judge them without the unknown true status of analyzed tags, given the too different interpretation of numeric values returned. In their Frequentist framework, the estimator pi = xi/mi is used for πi and a linear combination of these abundances is proposed as the correct way to combine results from different libraries: where wi are the weights that yield an unbiased minimum variance estimator Vu for weighted proportion's variance and θ = (α, β) are the Beta pdf parameters. However, this unbiased variance could be unrealistically small when it becomes smaller than the sampling variability. We know that the variance of this model cannot be smaller than the variance eventually obtained if we do not consider within-class variability. Therefore, they propose the final ad hoc estimator: V = max [Vu; Vpseudo-lib]     (14) where: The max(·) function assure that V is not unrealistic small when Vu is unrealistic small. To fit all these parameters, they used the computationally practical Method of Moments. Once pA, pB, VA and VB are found for classes A and B, these authors test if the proportions are significantly different proposing the use of a tw statistics as following a Student's tdf pdf: List of Abbreviations SAGE: Serial Analysis of Gene Expression MPSS: Massively Parallel Signature Sequencing EST: Expressed Sequence Tag pdf: probability density function GEO: Gene Expression Omnibus Authors' Contributions RV conceived and executed this work. HB helped with all biological issues. DFCP helped in differential expression detection methods and implemented the on-line web-based tool. CABP helped with Bayesian statistics and proposed the mixture ideas. Supplementary Material Additional File 1 Results for all evidence measures. This file allows the user to interactively define significance cutoffs for ranked tags. The ranks are based on evidence measures against "no differential expression" hypothesis, i.e., evidences closer to 0 (zero) denote higher confidence in differential expression and closer to 1 (one) denote no evidence of differential expression. Click here for file Acknowledgements RV is supported by FAPESP 02/04698-8 fellowship. We thank Tie Koide for critical reading of the manuscript and BIOINFO-USP/Rede-Vision for computational support. Figures and Tables Figure 1 The Bayes Error Rate illustration. This figure shows two illustrations of the proposed use of Bayes Error Rate E to define differentially expressing genes based on pdf of expression abundance π. The left example shows an obvious superposition of classes' pdf, thus a gene having this profile does not present evidence of differential expression between classes. The right example shows two pdfs "far apart" and genes with this kind of behaviour should be safely considered differentially expressing between two classes. Figure 2 Maximum a posteriori Beta pdfs of classes in main prototype cases. The predictive pdf and the Bayes Error Rate E of both tumoral (T) and normal (N) classes, for examples tags contained in the three important prototype cases described in text, are shown in the figure. The 'x' and 'o' marks represent observed abundances in each tumoral and normal. Frame a) shows case (i) example when methods agree with "no differential expression" conclusion. Frame b) shows case (ii) example when methods agree with "differential expression" conclusion. Frame c) and d) show case (iii) examples when classical P-value method leads to significant differential expression between classes and our method indicates pdf superposition if one take within-class variability into account. Individual observations indicate that the classes are not clearly divided, casting doubt on "differential expression" conclusion. Figure 3 Effect of "small" size libraries on the final result. The predictive pdf and the Bayes Error Rate E of both tumoral (T) and normal (N) classes for examples tags contained in the three important prototype cases described in text are shown. The 'x' and 'o' marks represent observed abundances in each tumoral and normal libraries. Frame a) shows the result using all libraries. The "small" libraries (size < 50,000) are highlighted with their counts over library size. Frame b) shows results excluding those "small" libraries. It is clear that results are pretty much the same and that "small" libraries are not (necessarily) outliers of the sampling. Figure 4 Qualitative comparison of Bayes Error Rate and t-test approximation. It is shown the Bayes Error Rate (E) versus the t-test approximation P-value (Baggerly) [12] for each tag. The red lines are arbitrary cutoffs that define significance regions E ≤ 0.1 and Baggerly ≤ 0.01. The green line is a LOWESS trend fit. Figure 5 Illustration of Maximum a Posteriori parameter estimation. The maximum a posteriori parameters of Eq.12 in an artificial example it is shown. The bi-dimensional pdf from which "hat" (pointed by the arrow) parameters are extracted is proportional to that described in Eq.8. Figure 6 Snapshot of the web-interface for our SAGEbetaBin method. An illustration of on-line tool implemented to make our method easily available it is shown. Researchers submit their data (A) and receive, by e-mail, an alert when we finished the job along with instructions to get results in a password-protected web-page (B). If the ID supplied is a human tag (C), then results are linked with SAGE genies' tag-to-gene map. The individual observations graphics, as utilized in this work, is available on-demand (D). Table 1 Brain tumor and normal libraries from SAGE Genie used as real data application. # Library Name GEO accession Total Tags 1 SAGE_Brain_astrocytoma_grade_III_B_H1020 GSM697 51573 2 SAGE_Brain_astrocytoma_grade_III_B_H970 GSM14763 106982 3 SAGE_Brain_astrocytoma_grade_III_B_R140 GSM14773 118733 4 SAGE_Brain_astrocytoma_grade_III_B_R927 GSM14766 107344 5 SAGE_Brain_normal_cerebellum_B_1 GSM761 50385 6 SAGE_Brain_normal_cerebellum_B_BB542 GSM695 40500 7 SAGE_Brain_normal_cortex_B_BB542 GSM676 94233 8 SAGE_Brain_normal_cortex_B_pool6 GSM763 62451 9 SAGE_Brain_normal_peds_cortex_B_H1571 GSM786 77554 10 SAGE_Brain_normal_substantia_nigra_B_1 GSM14796 42498 11 SAGE_Brain_normal_thalamus_B_1 GSM713 24015 ==== Refs Velculescu VE Zhang L Vogelstein B Kinzler KW Serial analysis of gene expression Science 1995 270 484 487 7570003 Margulies EH Kardia SL Innis JW Identification and prevention of a GC content bias in SAGE libraries Nucleic Acids Res 2001 29 e60 11410683 10.1093/nar/29.12.e60 Stern MD Anisimov SV Boheler KR Can transcriptome size be estimated from SAGE catalogs? Bioinformatics 2003 19 443 448 12611798 10.1093/bioinformatics/btg018 Stollberg J Urschitz J Urban Z Boyd CD A Quantitative Evaluation of SAGE Genome Research 2000 10 1241 1248 10958642 10.1101/gr.10.8.1241 Akmaev VR Wang CJ Correction of sequence based artifacts in serial analysis of gene expression Bioinformatics 2004 20 1254 1263 14871862 10.1093/bioinformatics/bth077 Morris JS Baggerly KA Coombes KR Bayesian shrinkage estimation of the relative abundance of mRNA transcipts using SAGE Biometrics 2003 59 476 486 14601748 10.1111/1541-0420.00057 Blades NJ Jones JB Kern SE Parmigiani G Denoising of data from serial analysis of gene expression Bioinformatics Brenner S Johnson M Bridgham J Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays Nature Biotechnology 2000 18 630 634 10835600 10.1038/76469 Man MZ Wang X Wang Y POWER_SAGE: comparing statistical tests for SAGE experiments Bioinfo matics 2000 16 953 959 10.1093/bioinformatics/16.11.953 Romualdi C Bortoluzzi S Danieli GA Detecting differentially expressed genes in multiple tag sampling experiments: comparative evaluation of statistical tests Hum Mol Genet 2001 19 2133 2141 10.1093/hmg/10.19.2133 Ruijter JM Kampen AHC Baas F Statistical evaluation of SAGE libraries: consequences for experimental design Physiol Genomics 2002 11 37 44 12407185 Baggerly KA Deng L Morris JS Aldaz CM Differential expression in SAGE: accounting for normal between-library variation Bioinformatics 2003 19 1477 1483 12912827 10.1093/bioinformatics/btg173 Aitchison J Dunsmore IR Statistical Prediction Analysis 1975 Cambridge: Cambridge University Press Bueno AMS Pereira CAB Rabello-Gay MN Stern JM Environmental genotoxicity evaluation: Bayesian approach for a mixture statistical model Stochastic Environmental Research and Risk Assessment 2002 16 267 278 10.1007/s00477-002-0100-x Duda RO Hart PE Stork DG Pattern Classification 2000 2 New York: Wiley-Interscience Press Vêncio RZN Brentani H Pereira CAB Using credibility intervals instead of hypothesis tests in SAGE analysis Bioinformatics 2003 19 2461 2464 14668232 10.1093/bioinformatics/btg357 Yeung KY Medvedociv M Bumgarner RE Clustering gene-expression data with repeated measurements Genome Biology 2003 4 R34.1 R34.17 12734014 SAGE Genie Home Page Boon K Osório EC Greenhut SF Schaefer CF Shoemaker J Polyak K Morin PJ Buetow KH Strausberg RL Souza SJ Riggins GJ An anatomy of normal and malignant gene expression Proc Natl Acad Sci USA 2002 99 11287 11292 12119410 10.1073/pnas.152324199 Audic S Claverie J The significance of digital gene expression profiles Genome Research 1997 7 986 995 9331369 Romualdi C Bortoluzzi S D'Alessi F Danieli GA IDEG6: a web tool for detection of differentially expressed genes in multiple tag sampling experiments Physiol Genomics 2003 12 159 162 12429865 IDEG6 Home Page SAGEbetaBin Home Page Lal A Lash AE Altschul SF Velculescu V Zhang L McLendon RE Marra MA Prange C Morin PJ Polyak K Papadopoulos N Vogelstein B Kinzler KW Strausberg RL Riggins GJ A public database for gene expression in human cancers Cancer Research 1999 21 5403 5407 Chen H Centola M Altschul SF Metzger H Characterization of gene expression in resting and activated mast cells J Exp Med 1998 188 1657 1668 9802978 10.1084/jem.188.9.1657 Aitchison J A General class of distributions on the simplex J R Statistical Soc B 1985 47 136 146 Ihaka R Gentleman R R: A language for data analysis and graphics Journal of Computational and Graphical Statistics 1996 5 299 314 R project Home Page Gene Expression Omnibus Home Page

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==== Front BMC BioinformaticsBMC Bioinformatics1471-2105BioMed Central London 1471-2105-5-1211534503310.1186/1471-2105-5-121Research ArticleSED, a normalization free method for DNA microarray data analysis Wang Huajun [email protected] Hui [email protected] Oscient Pharmaceuticals Corporation, 100 Beaver St, Waltham, Massachusetts 02453, USA2004 2 9 2004 5 121 121 26 4 2004 2 9 2004 Copyright © 2004 Wang and Huang; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Analysis of DNA microarray data usually begins with a normalization step where intensities of different arrays are adjusted to the same scale so that the intensity levels from different arrays can be compared with one other. Both simple total array intensity-based as well as more complex "local intensity level" dependent normalization methods have been developed, some of which are widely used. Much less developed methods for microarray data analysis include those that bypass the normalization step and therefore yield results that are not confounded by potential normalization errors. Results Instead of focusing on the raw intensity levels, we developed a new method for microarray data analysis that maps each gene's expression intensity level to a high dimensional space of SEDs (Signs of Expression Difference), the signs of the expression intensity difference between a given gene and every other gene on the array. Since SED are unchanged under any monotonic transformation of intensity levels, the SED based method is normalization free. When tested on a multi-class tumor classification problem, simple Naive Bayes and Nearest Neighbor methods using the SED approach gave results comparable with normalized intensity-based algorithms. Furthermore, a high percentage of classifiers based on a single gene's SED gave good classification results, suggesting that SED does capture essential information from the intensity levels. Conclusion The results of testing this new method on multi-class tumor classification problems suggests that the SED-based, normalization-free method of microarray data analysis is feasible and promising. ==== Body Background DNA microarray technology is now playing an increasingly important role in biomedical research. Microarray technology gives one the opportunity to measure gene expression levels of thousands to tens of thousands of genes simultaneously, in order to study the differential gene expression pattern between different developmental stages, diseases states and samples treated with drugs or other compounds. Before comparing data from different arrays to address these biological questions, however, a "much more mundane but indispensable " normalization step [1] is currently used in most microarray analyses. Because of the slight difference in RNA quantities, imaging settings and other variables, even in very controlled experiments the intensity levels from different arrays are of different scales and need to be normalized before they can be compared with each other. Various normalization methods have been developed and some are widely used. The simplest method is total intensity based normalization [2]; this approach scales intensity levels of every gene by a constant factor so that total intensities of all the arrays are the same. "Spiked-in" based normalization methods scale intensity based on spiked-in standards [3]. Nonlinear normalization methods use local regression to scale intensities to compensate for the intensity-dependent differences between arrays [4-6]. For most current applications, these normalization methods seem to be adequate. However, the residual left by a less than perfect normalization procedure is another source of non-biological variation that is usually non-desirable, especially when the differences in expression levels are expected to be small [7]. In addition, if the goal is meta-analysis of multiple sets of microarray data [8,9] systematic differences between experiments may result in a normalization artifact. We were therefore interested in developing an approach to analyse microarray data without first performing a normalization step. Our approach was partly inspired by non-parametric statistical methods [10]. For example, nonparametric methods that use ranks [11,12] to compare microarray results, in addition to being distribution free, have the additional advantage of being normalization free. DNA microarray technology has been used widely in biomedical studies. One interesting application is in the area of molecular classification; one popular use is in the comparison of tumor samples. Since clinical and histopathological classification is sometimes difficult and labor-intensive, the use of genome wide expression patterns to classify tumor samples has recently become a very active research area [13-16]. Although some tumors appear to be amenable to classification using microarray data [17,18], general multiple tumor classification using microarray data has proved to be an interesting and challenging task for several reasons: the general difficulties inherent in multi-class classification problems, the small number of samples available, and the inherent biological variation between specimens, etc. We decided to use multi-class tumor classification as a test case to illustrate the power of our approach. We compared our results for a multi-class tumor classification problem with more conventional approaches published by Ramaswamy et al. [19] and Yeang CH et al. [20]. These authors compared the accuracies of using k-Nearest Neighbors (kNN, 60–70%), Weighted Voting (WV, 60–70%) and Support Vector Machine (SVM, 80%) algorithms in a multi-class tumor classification problem and concluded that SVM is a more powerful machine learning algorithm for this application. Results Normalization Free approach to microarray data analysis Generally, measurements on single microarrays give a real-valued intensity level xi (1<= i <= N) for each gene i on the array, where N is the total number of genes on the array. Without first doing some type of normalization, the intensity level of gene i from array A, xiA, cannot be directly compared with the intensity level of gene i from array B, xiB. In this study, we sought an alternative quantity or quantities that can be directly compared between different arrays without compromising important biological information. One obvious candidate is ri, the rank of intensity level of gene i on the array. However, we felt that rank is not an adequate measure because information about relative expression level is not represented explicitly. Instead, we decided to use the following measures. Let sij = 1 if(xi - xj > 0) 0 otherwise     (1) , where 1<= i, j <= N. Basically, sij is the sign of the intensity difference of gene i and j on a single microarray and therefore will remain unchanged under any monotonic transformation of x. Therefore, instead of computing with the absolute expression level of a gene, its relative level to all the other genes on the microarray is used. For each gene i, instead of one real valued xi, the approach uses si = (si1, ..., sij, ..., siN), a binary vector of size N. For ease of reference, we will simply refer to this value as the SED (Signs of Expression Difference) of gene i; and the entire matrix (sij) the SED of the array. Given (xi), sij is simply and uniquely defined but (sij) does not uniquely determine xi so some information is lost by only using (sij) instead of (xi). Since ri = Σj = 1,...,N sij is the rank of gene i in terms of intensity levels, rank information is preserved in (sij). What is lost in the transformation from (xi) -> (sij) is just the intensity differences between the closest ranked genes, which in most cases are small, considering that microarray data are generally considered "very noisy". It was our major goal to demonstrate that (sij) has indeed captured important components of the information from (xi). Instead of directly using the intensity levels x, and its derivatives such as the mean μ, the standard deviation σ and the signal to noise ratio (S2N) between two sample groups A and B, [(μA + μB)/(σA+ σB)], we will use (sij) to compare gene expression differences between arrays. Since we expect measurement variations within an array will be less than those between arrays and we take the signs of relative expression differences to get SED, we expect the SED will be less "noisy". However, the value of any single sij may still vary between technical and biological replicates. One would expect more sij would change values randomly if the technical replicates was done on arrays that were fabricated in a different run than arrays from same run, for example. Biological variations are expected to be even more frequent. However, we hypothesize that we can perform statistical analysis on the SED, which contains tens of thousands of sij for a single gene, and minimize the impact of such noises. We will also consider (spij), a natural generalization of the SED concept. Here, spij is the probability of xi > xj. In other words, imagining one can get a large number, n, of either technical or biological replicates of the sample of interest, then spij = m/n as n -> 8, where m = ΣK = 1,...,n sijK and sK is for replicate K. We will call (spi) = (spi1, ..., spij, ..., spiN) the SED probabilities of gene i. Note that in calculating both SED and SED probabilities, only intensity comparisons within arrays are involved and therefore forego the normalization step. For example, if gene i is more highly expressed in sample A than B we would expect that more sijA than sijB would be 1 instead of 0 and the overall (very loosely defined) spijA would be larger than spijB. Since rank can be calculated from SED, any rank based method can be expanded to use SED. A gene i's SED can be viewed in two different perspectives. On one hand, it provides information about gene i's expression level relative to every other gene on the array, and therefore can be used to examine gene i's expression patterns between samples. On the other hand, it also provides information about the expression levels of all the other genes on the array, using the gene i as a control, in essence. Therefore, SED can be used to study questions either at the gene level or at the array level. In this paper, we focus on solving a simpler problem at the array level where it is not necessary to decide whether the expression level of an individual gene is increased or decreased between array A and B and by how much. Rather, it is focused on whether the overall expression patterns are different at all between array A and B. Multi-class classification of tumor samples To test whether (sij) and (spij) extracts most of the information from (xi), we used these values in a test case of a multi class classification problem described by Ramaswamy et al. and Yeang et al [19,20]. Two algorithms were used to classify each of the 144 tumor samples into one of 14 tumor classes. One is the Naive Bayes (NB) classifier [21] using SED probabilities. The other is the Nearest Neighbor (NN) classifier using SED. In the NB method, to classify a sample T, we first calculate (spijC) for each class C (1 <= C <= 14) using training samples, i.e. the 144 samples with T taken away (for details see methods). Then sample T is classified according to: score(T, C)= Σi = 1,...N Σj = 1,...,N log(pij),     (2) where pij = spijC if sijT = 1 1 - spijC otherwise T is simply classified to the class C that has the maximum score. In the NN method, we compute instead, for each training sample t, matches(T, t) = Σi = 1,...N Σj = 1,...,N δ(sijT, sijt),     (3) where δ(x, y) = 1 if x = y. 0 otherwise Then T is classified to class C of the sample t that has the maximum matches. If one is to give a statistical interpretation of these scores, one can simply view (spij) as defining a multi-binomial probability model. In addition, one could consider each sij as a draw from a binomial distribution with probability pij = spij. Then, the score(T, C) is simply a logarithm of the probability p to get all the sij exactly the same as sijT under the probability model C where pij = spijC. (Since each class defines a different probability model, score(T,C) for difference class C, in theory, should not be directly compared. Instead, a P value should be calculated from the probability model for Pr(p<exp(Score(T, C))) and used to evaluate the closeness of sample T to each class C. For simplicity, we are not considering such issues here.) When the NB algorithm was applied to the 144 samples, the accuracy obtained was about 63%; the NN algorithm performed slightly better and gave an accuracy of 70%. Feature selection Depending on the algorithm, a better classification result can sometimes be obtained by using a subset of genes [22,23]. We were interested to know whether feature selection helps to increase accuracy in our approach. Within our framework, it is easier to treat (i,j) pairs as selection units. We therefore filtered out (i,j) pairs where the variance of spij across the 14 tumor classes was less than a pre-determined value and left the rest of the algorithm unchanged. We reasoned that the filtered-out part of the matrix has less discriminating power across the tumor spectrum and might add noise due to the small sample sizes used. Using the NB algorithm, the best result achieved was 70% with a cutoff σ2 = 0.06, while the NN method with σ2 = 0.05 gave 77%. Table 1 lists the accuracies achieved using different feature cutoffs. "Single gene's SED" based classifier The above procedure utilized the entire SED matrix as a classifier. In other words, all the relations between genes were considered in the classification. To determine whether the inclusion of the whole matrix was actually required to achieve the current accuracy, we investigated the efficacy of using single gene's SED as classifiers. In these cases, we define a classifier based on gene i and its relative expression to every other gene. Therefore, the scorei(T, C) = Σj = 1,...,N log(pij), where pij is as same as mentioned previously. Similarly, matchesi(T, t) = Σj = 1,...,N δ(sijT, sijt) defines a classifier based on gene i's SED. Fig. 1 shows a display of the cumulative frequency of single gene SED based classifiers versus accuracy for all the 16063 classifiers. In general, single gene-based classifiers performed worse than the whole genome-based classifiers, as expected. Nevertheless, most of the classifiers performed reasonably well, compared with just using single gene expression levels. About 80% of the single gene based classifiers resulted in an accuracy between 40% and 60%, while about half of the classifiers had an accuracy greater than 50%. These results suggest that there is a lot of redundant information in the SEDs and SED probabilities and that our method should be reasonably robust. We then investigated the number of genes that are required to achieve the current accuracy. Fig. 2 shows the combined results for classifiers using only a subset of genes. Our results suggest that a subset of genes (~200) is sufficient for predictions and that the prediction accuracy is stable after 1000 genes. Different classification accuracy between tumor classes From the analyses described above, we noticed that there was a significant difference in accuracy between different tumor classes. For 3 classes (LY, LE, CNS) we obtained either 100% or close to 100% accuracy (see Table 2 for detail). Since these happen to correspond to the 3 classes with more than double the number of training samples than the other classes, we tested whether this high accuracy is due to the larger sample sizes by using only 8 training samples for every tumor classes. The results were essentially the same, indicating that sample size is not the issue. On the other hand, there are classes where we obtained very poor results; these often happen to be the same classes where SVM in [19,20] performed poorly as well (see Table 2). We were interested in exploring the possible reasons for misclassification. In Fig. 3, a scatterplot of the SED Match scores (without feature selection) between 8 OV samples and 8 CNS samples is displayed. The OV and CNS class were selected since one is "very hard" and the other is "very easy" to classify. Without trying to be statistically correct, the plot does suggest that samples of the OV class are in general "farther away" from each other, compared with those from the CNS class. As this may be one of the reasons that the OV class is harder to classify, algorithms that take this kind of information into account may perform better than the simple ones we have presented here. Discussion Although we used the multi-class tumor classification problem as our test case, our major goal was to illustrate the feasibility of the normalization free SED approach, and not in sample classification per se. Therefore, we chose the algorithms NB and NN for their simplicity and not for their performance in solving this specific problem. The performance of a classifier depends, in this case, mainly on the power of its algorithm, and the data representation it used. From a machine learning perspective, one can simply view the intensity -> SED transformation as a change in data representation, a mapping from the gene's attribute, intensity x, to some features SED. It was our goal to demonstrate that the new features (SED and SED probability), in addition to being normalization free, still convey the essential information in the original attribute, the intensity x. Since the data representation is quite different between the intensity x (a real valued quantity) and SED (a binary valued vector of rather large size N), it is difficult to directly compare the two. No obvious yet non-trivial algorithms work with both representations; even if there were such an algorithm it is not clear that it would be the right one to use for comparison as it might well be the case that different data representation works best with different algorithms. Here, we have limited the presentation to some empirical results with SED representation, which are comparable with results using several different algorithms that are based directly on raw intensity [19,20]. Our classification results are close to those obtained with WV and kNN methods, which are based on directly focusing on intensity levels. Previous results using SVM were significantly better, but we feel the differences are due more to the power of the algorithm [24] than the way information is coded. In fact, slightly more accurate results are obtained with modification of algorithms that directly manipulate intensity levels [25,26]. We do not imply that the algorithms (NB and NN) we chose are better than other alternatives (and we do not have empirical evidence pointing either way). Instead, we fully expect more sophisticated algorithms would work better with the SED approach as well. Certainly, SED probability is more information rich than SED. We expect that an SED probability based analysis would perform better than the simple binary valued SED. In this paper, we mainly tested the SED. SED probability is only used for a group of samples, not for single samples. If one limits oneself to use only raw data, then for single arrays one can only get SED. However, if some assumptions about the patterns of gene expression levels can be made, one can certainly get an estimation of SED probability even for a single array. For example, as in some nonlinear normalization algorithms, if one assumes that the variation of expression levels are similar for genes with similar expression levels, then one can estimate SED probability from a probability model. Also, the magnitude of the intensity difference can also be used to help such an estimation. Alternatively, as more and more microarray data become available, one can use other similar samples to get an estimation of a prior SED probability, and then use a Bayesian approach to estimate the sample's SED probability. The obvious disadvantage of our SED based approach is that for each gene expression level, one is not dealing with a single real number but instead a vector of size N, where N is in the tens of thousands. This could significantly increase both computing time and memory requirement (however, see methods for details) On the other hand, it also has certain advantages: 1) It is free of normalization noise. Since it is generally believed that biological variation is larger than technical variation and normalization noise is just another source of technical variation, the benefit here is only of a limited scope. However, it may be important when the expression level difference one is interested in is small. 2) In addition to being normalization free, SED and SED probability also have the advantage in being distribution free, and therefore could perform better if the intensity levels were non-normal. 3) SED and SED probability are easier to interpret. SED values can easily be checked against raw intensity levels according to Eq. (1). While SED probability is one step further away from intensity levels, one could still have an intuitive sense of it and make comparisons between different experiments. It would be much harder to have a real grasp of the absolute gene expression level, except that it is "high" or "low" or somewhere "in between"; it is certainly harder to compare between experiments intuitively. We have only tested the SED approach on datasets that are from the same chip format. Data from different chip formats or complete different technology platforms, of course, would be harder to compare. But they are also challenge for normalization based method. It would certainly be interesting to compare SED and normalization based method under these more challenging conditions. If this normalization-free approach (SED) proves to retain the essential biological information in general, its application may be extended to meta-analysis where different datasets could be integrated and intervalidated. The method could also be used when the number of arrays is a limiting factor for experiments. For example, one could take advantage of the massive amount of public array data, obtain prior distribution of SED probabilities from datasets with similar conditions, and analyze new data within a Bayesian framework. If the performance of the nearest neighbor method in general is anywhere close to what we demonstrated in the multi tumor classifications here (as is clear from Eq. (3) and Fig. 3, the nearest neighbor method, without feature selection at least, allows direct sample vs. sample comparison. Note also that the samples in the multi tumor problems are from different biological specimens, therefore, large between-sample-variation is expected), it might be used as a microarray database query method, i.e., to find similar microarray results in the database that are "similar" to one's own, independent of array annotations. It might also be worth noting that the SED approach could easily be applied to other kinds of comparative data analysis for samples with very large numbers of "noisy" attributes. The SED approach may also perform better when between-sample-variation is large, especially if such variation contains some rather uninteresting technical measurement errors that would not affect within-sample-variations. Conclusions We have proposed a new approach to analyze microarray data and tested the method on a set of publicly available datasets. The results were comparable to those obtained with some widely used normalization based algorithms. We hope that we have demonstrated that this normalization free method is feasible and promising. We think the SED based, normalization free approach could be used to complement the more popular normalization based approaches in microarray data analysis. Methods Microarray data for multiple tumor samples were downloaded from . Naive Bayes and Nearest Neighbour Classifiers were implemented in the Java programming language. Ad hoc analysis was done with perl scripts. Graphics were generated using the R computing environment. Naive Bayes method Because of the uneven and relatively small sample sizes for each tumor class (mostly 8 but up to 24), extra care was taken in computing spij. Assuming a prior probability of 0.5, spij was estimated by Bayesian posterior probability (m+1)/(n+2) where n is the total number of samples in the class and m is the total number of samples where xi > xj. For classes that were over-represented (sample size > 8), the threshold of spij was set to [0.125, 0.875], since the NB method is sensitive to the extreme values of sp, and samples can be over-predicated without thresholds. In addition, several alternatives were tested to demonstrate that our results were reasonably robust and not sensitive to the particular choices we made: 1) To examine the influence of the sample size, in a separate analysis the sample size of ME, LE, CNS class was artificially reduced to 8, i.e. only the first 8 samples were used to calculate spij with no significant change of results observed; 2) Since spij depends on the sample size n for each tumor class, we have applied a "sample replacement" strategy in addition to the usual "take-one-sample-out" approach for cross-validation, i.e. when one sample is taken out as the test sample, another sample from the same class is duplicated to take its place to keep the sample size constant. Essentially the same results were obtained. Results reported are from the sample replacement runs. In Feature Filtering, the variance of spij between all 14 classes was calculated as: σ2 = (ΣC = 1,...,14,spijC * spijC)/14 - ((ΣC = 1,...,14 spijC)/14)2     (4) with the test sample taken out, and used as the criterion for feature exclusion. Nearest neighbor method Feature filter was done as in the Naive Bayes Method. Software implementation and availability The analysis was done on a computer (Pentium M 1.5 GHz) operating under Microsoft XP. Both Naïve Bayes and Nearest Neighbor Classifiers are implemented in Java. Since SED can be easily calculated from the raw intensities only the later are kept in memory and SED are computed from the intensities on an as-needed bases. Memory needed to analysis the 144 samples is less than 64 MB. The most computationally intensive algorithm that we tried is the Nearest Neighbor method without any feature selections and it takes about 10 sec to calculate SED score for one pair of tumor samples with about 16000 genes. A Java program named SED (including source code) to perform nearest neighbor analysis of microarray samples is freely available by contacting author at [email protected]. Authors' contributions H.W. conceived of the SED study and performed implementations. H.H. refined the approach and provided additional statistical insight on SED. Both authors read and approved the final manuscript. Acknowledgements The authors wish to thank Dr. Randall D. Little for his careful reading and extensive editing of the manuscript. This work is supported by grant R44 AG022242-01 from NIH National Institute of Aging of the National Institutes of Health, USA Figures and Tables Figure 1 Cumulative Frequency of Single Gene SED classifiers versus cross validation accuracy. The X axis represents the cross validation accuracy and the Y axis represents the cumulative frequency of the single gene based classifiers. For each point (x,y) on the curve, y equals the percentage of classifiers that have an accuracy less than or equal to x. Solid curve – NN method with cutoff σ2 = 0.05. Dashed line – NB method with σ2 = 0.06. Figure 2 Evaluation of the number of genes required to achieve the highest accuracy. The X axis represents the number of genes used in the classifiers. The Y axis represents the cross validation accuracy. Solid line – NN method with cutoff σ2 = 0.05. Dashed line – NB method with σ2 = 0.06. X is in log scale. As X increases more genes are included in the classifier in the order they are represented in the original microarray (unselected for performance). Figure 3 Scatterplot of SED Match scores of OV and CNS samples. The X axis represents the SED Match scores from eq. (3) / N2, where N = 16063, the total number of genes. The Y axis represents the sample id. Samples 1–8 (shown in red) are from class OV while 9–16 (shown in blue) are from class CNS. Only 8 samples from the CNS class were used for ease of comparison. Other samples gave similar results. For each sample, its Match scores / N2 against all the other 7 samples within the class are shown. The Match scores of OV class are much more dispersed (to the left), compared with that of CNS class. Table 1 The relationship between accuracy (%) and the filter threshold The cross validation accuracies with Naive Bayes (NB) and Nearest Neighbor (NN) Methods are displayed with different filter cutoffs. The percentages of the features (gene pairs) used are listed as well. Since the number of features used are slightly different for different test samples, ranges are shown. Filter Threshold No Filter 0.02 0.04 0.05 0.06 0.08 NB 63 66 68 69 70 68 NN 70 73 75 77 75 74 Features used (%) 100 41–43 12–13 4.9–5.9 1.7–2.1 0.12–0.17 Table 2 Summary of cross validation results. All 14 tumor types and their abbreviations are listed in the first column. The sample sizes for each tumor type and the number of successfully classified samples by the SVM algorithm (from ref. [19]), the NB (Naive Bayes) algorithm with cutoff σ2 = 0.06 and the NN (Nearest Neighbor) algorithm with σ2 = 0.05 are listed. Tumor type (Abbreviation) Sample Size SVM [19] NB NN Ovary (OV) 8 3 1 4 Lung (LU) 8 4 1 3 Bladder (BL) 8 5 7 5 Melanoma (ML) 8 5 6 6 Renal (RE) 8 5 5 6 Pancreas (PA) 8 5 6 6 Colorectal (CR) 8 6 1 3 Prostate (PR) 8 6 6 6 Breast (BR) 8 7 3 4 Uterus (UT) 8 7 6 6 Mesothelioma (ME) 8 8 6 6 Lymphoma (LY) 16 16 14 16 Central Nervous System (CNS) 16 16 16 16 Leukemia (LE) 24 24 23 24 ==== Refs Quackenbush J Microarray data normalization and transformation Nat Genet 2002 32 496 1 12454644 10.1038/ng1032 Affymetrix Affymetrix GeneChip Expression Analysis Technical Manual 2003 Affymetrix Inc., Santa Clar, CA Hill AA Brown EL Whitley MZ Tucker-Kellogg G Hunter CP Slonim DK Evaluation of normalization procedures for oligonucleotide array data based on spiked cRNA controls Genome Biol 2001 2 RESEARCH0055 11790258 10.1186/gb-2001-2-12-research0055 Bolstad BM Irizarry RA Astrand M Speed TP A comparison of normalization methods for high density oligonucleotide array data based on variance and bias Bioinformatics 2003 19 185 93 12538238 10.1093/bioinformatics/19.2.185 Tseng GC Oh MK Rohlin L Liao JC Wong WH Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects Nucleic Acids Res 2001 29 2549 57 11410663 10.1093/nar/29.12.2549 Yang YH Dudoit S Luu P Lin DM Peng V Ngai J Speed TP Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation Nucleic Acids Res 2002 30 e15 11842121 10.1093/nar/30.4.e15 Mootha VK Lindgren CM Eriksson KF Subramanian A Sihag S Lehar J Puigserver P Carlsson E Ridderstrale M Laurila E Houstis N Daly MJ Patterson N Mesirov JP Golub TR Tamayo P Spiegelman B Lander ES Hirschhorn JN Altshuler D Groop LC PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes Nat Genet 2003 34 267 73 12808457 10.1038/ng1180 Moreau Y Aerts S De Moor B De Strooper B Dabrowski M Comparison and meta-analysis of microarray data: from the bench to the computer desk Trends Genet 2003 19 570 7 14550631 10.1016/j.tig.2003.08.006 Rhodes DR Barrette TR Rubin MA Ghosh D Chinnaiyan AM Meta-analysis of microarrays: interstudy validation of gene expression profiles reveals pathway dysregulation in prostate cancer Cancer Res 2002 62 4427 33 12154050 Hollander M Wolfe DA Nonparametric Statistical Methods 1999 2 John Wiley & Sons, New York Troyanskaya OG Garber ME Brown PO Botstein D Altman RB Nonparametric methods for identifying differentially expressed genes in microarray data Bioinformatics 2002 18 1454 61 12424116 10.1093/bioinformatics/18.11.1454 Liu WM Mei R Di X Ryder TB Hubbell E Dee S Webster TA Harrington CA Ho MH Baid J Smeekens SP Analysis of high density expression microarrays with signed-rank call algorithms Bioinformatics 2002 18 1593 9 12490443 10.1093/bioinformatics/18.12.1593 Cleator S Ashworth A Molecular profiling of breast cancer: clinical implications Br J Cancer 2004 90 1120 4 15026788 10.1038/sj.bjc.6601667 Fu LM Fu-Liu CS Multi-class cancer subtype classification based on gene expression signatures with reliability analysis FEBS Lett 2004 561 186 90 15013775 10.1016/S0014-5793(04)00175-9 Meyerson M Franklin WA Kelley MJ Molecular classification and molecular genetics of human lung cancers Semin Oncol 2004 31 4 19 14981576 Bertucci F Salas S Eysteries S Nasser V Finetti P Ginestier C Charafe-Jauffret E Loriod B Bachelart L Montfort J Victorero G Viret F Ollendorff V Fert V Giovaninni M Delpero JR Nguyen C Viens P Monges G Birnbaum D Houlgatte R Gene expression profiling of colon cancer by DNA microarrays and correlation with histoclinical parameters Oncogene 2004 23 1377 91 14973550 10.1038/sj.onc.1207262 Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson J JrLu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PO Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling Nature 2000 403 503 11 10676951 10.1038/35000501 Golub TR Slonim DK Tamayo P Huard C Gaasenbeek M Mesirov JP Coller H Loh ML Downing JR Caligiuri MA Bloomfield CD Lander ES Molecular classification of cancer: class discovery and class prediction by gene expression monitoring Science 1999 286 531 7 10521349 10.1126/science.286.5439.531 Ramaswamy S Tamayo P Rifkin R Mukherjee S Yeang CH Angelo M Ladd C Reich M Latulippe E Mesirov JP Poggio T Gerald W Loda M Lander ES Golub TR Multiclass cancer diagnosis using tumor gene expression signatures Proc Natl Acad Sci U S A 2001 98 15149 54 11742071 10.1073/pnas.211566398 Yeang CH Ramaswamy S Tamayo P Mukherjee S Rifkin RM Angelo M Reich M Lander E Mesirov J Golub T Molecular classification of multiple tumor types Bioinformatics 2001 17 S316 22 11473023 Mitchell T Machine Learning McGraw-Hill 1997 Hastie T Tibshirani R Eisen MB Alizadeh A Levy R Staudt L Chan WC Botstein D Brown P 'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns Genome Biol 2000 1 RESEARCH0003 11178228 10.1186/gb-2000-1-2-research0003 Lyons-Weiler J Patel S Bhattacharya S A classification-based machine learning approach for the analysis of genome-wide expression data Genome Res 2003 13 503 12 12618382 10.1101/gr.104003 Vapnik V Statistical learning theory 1998 John Wiley & Sons, New York Shedden KA Taylor JM Giordano TJ Kuick R Misek DE Rennert G Schwartz DR Gruber SB Logsdon C Simeone D Kardia SL Greenson JK Cho KR Beer DG Fearon ER Hanash S Accurate molecular classification of human cancers based on gene expression using a simple classifier with a pathological tree-based framework Am J Pathol 2003 163 1985 95 14578198 Peng S Xu Q Ling XB Peng X Du W Chen L Molecular classification of cancer types from microarray data using the combination of genetic algorithms and support vector machines FEBS Lett 2003 555 358 62 14644442 10.1016/S0014-5793(03)01275-4

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==== Front BMC GenomicsBMC Genomics1471-2164BioMed Central London 1471-2164-5-591533102110.1186/1471-2164-5-59Research ArticleCombined analysis of expression data and transcription factor binding sites in the yeast genome Nagaraj Vijayalakshmi H [email protected]'Flanagan Ruadhan A [email protected] Adrian R [email protected] Jonathan R [email protected] Andrew K [email protected] Anirvan M [email protected] BioMaPS Institute, Rutgers University, Piscataway, NJ 08854, USA2 Department of Physics and Astronomy, Rutgers University, Piscataway, NJ 08854, USA3 Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA4 Medical Sciences Center, University of Wisconsin, Madison, WI 53706, USA2004 26 8 2004 5 59 59 30 4 2004 26 8 2004 Copyright © 2004 Nagaraj et al; licensee BioMed Central Ltd.2004Nagaraj et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The analysis of gene expression using DNA microarrays provides genome wide profiles of the genes controlled by the presence or absence of a specific transcription factor. However, the question arises of whether a change in the level of transcription of a specific gene is caused by the transcription factor acting directly at the promoter of the gene or through regulation of other transcription factors working at the promoter. Results To address this problem we have devised a computational method that combines microarray expression and site preference data. We have tested this approach by identifying functional targets of the a1-α2 complex, which represses haploid-specific genes in the yeast Saccharomyces cerevisiae. Our analysis identified many known or suspected haploid-specific genes that are direct targets of the a1-α2 complex, as well as a number of previously uncharacterized targets. We were also able to identify a number of haploid-specific genes which do not appear to be direct targets of the a1-α2 complex, as well as a1-α2 target sites that do not repress transcription of nearby genes. Our method has a much lower false positive rate when compared to some of the conventional bioinformatic approaches. Conclusions These findings show advantages of combining these two forms of data to investigate the mechanism of co-regulation of specific sets of genes. ==== Body Background A bioinformatic approach to identifying cis-regulatory elements controlling transcription has become feasible with the availability of complete genome sequences and large scale expression data using high-throughput methods such as microarrays [1,2] and SAGE [3]. The expression data provides a list of genes whose expression is significantly modified under a particular condition. However, this data does not indicate whether these genes are direct targets of a particular transcription factor or if the changes in expression are the result of an indirect effect caused by altering the expression of other transcription factors that work directly at the promoter. Using information about sequence preference for binding of particular transcription factors, one can identify possible regulatory binding sites within a sequenced genome. However, this approach does not indicate if the sites are functional. We have therefore developed an algorithm that combines both of these approaches to distinguish between the direct and indirect targets that are regulated by a particular transcription factor. We have applied this methodology to study the transcriptional regulatory system that specifies cell mating-type in the yeast Saccharomyces cerevisiae [4]. Yeast have three cell types, haploid a and α cells, and the a/α diploid, that differ in their ability to mate and in the proteins they express. Cell mating-type is determined in part by α2 and a1, which are cell-type-specific proteins that are members of the homeodomain (HD) DNA-binding family. In an a/α diploid cell, α2 binds with a1 to form a heterodimer complex that represses transcription of haploid-specific genes [5]. The crystal structures of the α2 HD binding DNA alone and in complex with a1 have been solved, providing models for how these complexes bind DNA [6,7]. Biochemical and mutational analysis of each protein and their DNA-binding sites have defined the requirements for DNA recognition by this complex [8-10]. Genome-wide expression analysis has also been performed on each of the different cell types [11]. The combination of these resources has allowed us to develop and test algorithms to identify target sites for the a1-α2 complex. Previous work, using a relatively simple binding site search program identified targets for the α2-Mcm1 complex, which represses a-cell-type specific genes in α and a/α cells [12]. The more advanced methods described in this paper have helped identify several novel targets of the a1-α2 complex that may be involved in cell-type specific processes. Interestingly, we identified several genes that are repressed in diploid cells but do not appear to be direct targets of the a1-α2 complex, suggesting that these genes are controlled by another transcriptional regulatory factor that is directly or indirectly regulated by the a1-α2 complex. We have also identified a number of a1-α2 target sites that do not repress adjacent genes. The combination of site preference and expression data is therefore a valuable tool to identify direct functional targets of a transcription factor or complex. Results Development of a search algorithm for targets of the a1-α2 complex To generate an algorithm that combines microarray expression data and mutational analysis of binding sites, we first defined a scoring method that ranks gene expression data. We utilized the microarray expression data from Galitski and coworkers for gene expression in the a and α haploid and a/α diploid cells, as well as various polyploids [11]. Since the a1-α2 complex should be absent in any of the homozygous a or α type polyploids (a, aa, ..., α,αα,..., etc.) we expect the expression of haploid-specific genes in these cells to be much higher than in cells that are heterozygous for the MAT locus (aα, aaα, aαα, aaαα, etc.). Thus, one term in the scoring function rewards lower expression in heterozygous cell types compared to the homozygous cell types (see the Methods section for details). We expect that most of the haploid-specific genes will be expressed equally in both of a and α cell types. Consequently, we have introduced a second term in the scoring function that penalizes such differences in expression in the two haploid cell types. This scoring function would identify haploid-specific genes that are repressed in diploid cells, but would not indicate if these genes are direct targets of the a1-α2 repressor complex. To identify genes from this ranking that are directly repressed by the a1-α2 complex we used the available mutational data on the a1-α2-binding site [9]. In these experiments, the effects of single base pair mutations of the a1-α2 consensus binding site were measured by assaying their ability to repress transcription of a heterologous promoter and by electrophoretic mobility shift DNA-binding assays (EMSA). Under the assumption that the level of expression is proportional to how often that site is unoccupied, we used the effects of the single base mutations to estimate the parameters for the binding energy of sites with different bases at each position. We then used this information to search for potentially strong binding sites in the promoter regions (in practice, 800 bp upstream of the translation start site) of every gene in the genome. This search provides us with a list of genes with putative a1-α2 binding sites in the promoter, irrespective of functionality. Even under ideal conditions, either of the above lists of genes would not directly indicate a haploid-specific gene directly repressed by the a1-α2 complex. In addition, our accuracy is limited by the noise in the microarray expression data, as well as by simplifying assumptions made to utilize single-base mutational data to score arbitrary sequences. We therefore decided to rank the genes using a scoring system that takes into account expression patterns across different mating types, as well as the likelihood of finding a good a1-α2-binding site in the promoter region of the gene. To test the significance of these composite scores, we generated permuted data, which combined random promoters with the expression data. This analysis suggested that the top 10–15 predictions were significant. The results of experiments on the 24 genes with the highest combined scores from our analysis are shown in Table 1. Table 1 Potential a1-α2 Binding Sites in Haploid-specific Genes ORF Gene Expression p-vala Binding p-valb Combined p-val a1-α2 ChIPc YDL227C HO 0.0006 0.0017 1.1e-6 +f YLR265C NEJ1 0.0003 0.0053 1.7e-6 + YBL016W FUS3 0.0001 0.0991 1.6e-5+ YOR212W STE4 0.0020 0.0082 1.7e-5 + YJR086W STE18 0.0008 0.0218 1.7e-5 + YHR005C GPA1 0.0005 0.0437 2.1e-5 + YDR103W STE5 0.0017 0.0298 5.2e-5 + YBR073W RDH54 0.0053 0.0116 6.2e-5 + YGR044C RME1 0.0009 0.0720 6.8e-5 +f YGL248W PDE1 0.0182 0.0040 7.3e-5 + YPL038W MET31 0.0292 0.0027 8.0e-5 + YDR088C SLU7 0.0303 0.0038 1.2e-4 - YGL052W 0.0117 0.0109 1.3e-4 - YJL157C FAR1 0.0013 0.1141 1.4e-4 + YPR122W AXL1 0.0091 0.0163 1.5e-4 +f YIL099W SGA1 0.0063 0.0267 1.7e-4 - YLR233C EST1 0.0226 0.0090 2.0e-4 - YKL182W FAS1 0.0578 0.0035 2.1e-4 - YMR053C STB2 0.0028 0.0884 2.5e-4 - YNL319W 0.0123 0.0222 2.7e-4 - YFR012W 0.0088 0.0125 2.8e-4 - YNL188W KAR1 0.0019 0.1890 3.6e-4 - YGL193C 0.0014 0.2654 3.8e-4 - YMR157C FMP39 0.1557 0.0026 4.0e-4 - YBR158Wd AMN1 0.0037 0.0221 8.7e-5 + YCL066We MATα1 0.1630 0.9510 1.5e-1 +f a The ranking of haploid-specific gene expression determined by analysis of microarray data [11]. b The ranking of potential a1-α2 target of haploid-specific genes. c A + indicates that the a1-α2 binds to the promoter by ChIP assay. d Identified in a search for sites with 1500 bp from the start of the ORF e Identified if remove the penalty for expression in one haploid cell type but not the other. f Identified as direct targets of the a1-α2 repressor complex in previous studies. a1-α2 binding to the promoter regions of genes identified in the search To evaluate the success of our computational algorithm for identifying direct targets of the a1-α2 repressor complex, we assayed binding by the complex to the identified promoters using chromatin-immunoprecipitation (ChIP) assays with polyclonal antibody directed against the α2 protein. In α haploid and a/α-diploid cells the α2 protein combines with the MADS-box transcription factor Mcm1 to bind to elements in the promoters of a-specific genes to repress their transcription [13,14]. We therefore included in each PCR a primer set for the promoter region of the a-specific gene STE6 to serve as a positive control for the ability to ChIP α2 in both haploid α and diploid a/α cells. This primer set also allowed us to rule out the possibility that the predicted a1-α2 target genes were immunoprecipitating because of binding by the α2-Mcm1 complex. The primer set for the YDL223C promoter, a gene not bound or repressed by the a1-α2 or α2-Mcm1 complexes, was included in the reaction as a negative control for non-specific immunoprecipitation of the DNA. A gel displaying the results for a few of the promoters that were assayed is shown in Figure 1 and the data summarized for all the promoters that were predicted to be directly repressed by the a1-α2 complex is listed in Table 1. In general, there is a very good correlation between the experimental data with the predictions based on our computational algorithm. Almost all of the high scoring genes were bound by the a1-α2 complex in vivo. Genes that had a combined p-value higher than the threshold of 1.5 × 10-4 (which corresponds to choosing the top 15 of the list in Table 1) do not appear to be strongly bound by the complex. This p-value corresponds to roughly a probability of one in six thousand, indicating it is possible to get such combination by chance. Figure 1 ChIP assays of promoter fragments that are predicted to be targets of the a1-α2 complex. ChIP assays with antibody to the α2 protein were performed on lysates from MATa/MATα (a/α) and mataΔ/MATα(Δ/α) cells. Total chromatin (TC) and immunoprecipitated (IP) samples were subjected to multiplex PCR with primers flanking potential a1-α2 sites (labeled hsg) in the indicated promoters. Primers for the promoter region of STE6, an a-specific gene that is repressed by the α2-Mcm1 complex in both cell types were used as a positive control for the ChIPs. Primers that hybridize in YDL223C, a gene that is not regulated by α2, was used as the negative control. The presence of a band over background levels from the test promoter from a/α lysates but not matΔ/α indicates that the a1-α2 complex is specifically binding to the promoter. Assays for the GPA1, FAR1, NEJ1, RDH54, MET31, PDE1, AMN1/CST13 and KAR1 are shown. A summary of the all of the ChIPs performed is in Table 1. In comparison to higher eukaryotes, most yeast promoters are relatively small and contain activator or repressor binding sites within several hundred base pairs of the start site of the open reading frame (ORF) of the gene. However, there are a few genes, like HO, whose regulation is controlled by a region several Kb long. Consequently, we did a separate search looking for additional a1-α2 binding sites that are within a region 1.5 Kb upstream of the ORF. Most of the sites identified in this search were well above the threshold value and were not bound by the a1-α2 complex in ChIP assays (data not shown). However, the search identified one site upstream of the AMN1/CST13 gene that was a potential target site. The ChIP analysis verified this as a functional target site for the a1-α2 complex in vivo (Figure 1, Table 1). Analysis of haploid-specific genes predicted not to be bound by a1-α2 Among the top 35 genes in the list ranked by haploid-specific expression score, more than half do not appear to contain an identifiable a1-α2 site by our analysis (lack of a significant binding site being defined as binding p-value greater than 2 × 10-4) (Table 2). Although there were no apparent strong affinity a1-α2-binding sites in the promoters of these genes, it is possible that there are several weak affinity sites in the promoter that were not identified in the search. If present, these sites may work cooperatively to increase binding by the a1-α2 complex to the promoter and therefore repress transcription. In support of this model we have found that under some conditions weak affinity a1-α2 sites in the HO promoter have a role in repression of the promoter (Mathias and Vershon, unpublished). Promoters with a number of weak affinity sites may therefore be directly regulated by the a1-α2 complex. However, it is also possible that these genes are indirectly repressed by a1-α2, through its ability to repress expression of another transcription factor that is required for expression of the genes identified in the microarray. To distinguish between these possibilities we assayed for a1-α2 binding to these promoters by ChIP (Figure 2 and Table 2). As predicted from the site identification analysis, the a1-α2 complex did not appear to bind to most of these promoters. This result suggests that these genes are not directly repressed by the complex. The one exception to our predictions was that the a1-α2 complex appeared to weakly bind to the NEM1 promoter in the ChIP assays (Figure 2). Interestingly, NEM1 is downstream of GPA1, a gene that is strongly repressed by the a1-α2 complex (Fig 1). Binding and repression by a1-α2 at GPA1 may help binding to weak sites in the NEM1 promoter. Alternatively, although we sheared the DNA used in the ChIP to an average of less than 500 bp, it is possible that there may have been some fragments that spanned the ~2 kb between the genes, thereby giving a positive result in the ChIP assay. Table 2 Haploid-specific Genes that Do Not Contain a1-α2 Target Sites ORF Gene Expression p-vala Binding p-valb Combined p-val a1-α2 ChIPc YIL117C PRM5 0.0011 0.3690 0.0004 - YLR159W 0.0015 0.4050 0.0006 - YBR051W 0.0022 0.4390 0.0010 - YLR080W EMP46 0.0024 0.6757 0.0016 - YIR039C YPS6 0.0025 0.7843 0.0020 - YCL014W BUD3 0.0027 0.3475 0.0009 - YPL189W GUP2 0.0030 0.3708 0.0011 - YJL077C ICS3 0.0032 0.9540 0.0030 - YML042W CAT2 0.0033 0.7564 0.0025 - YHR004C NEM1 0.0035 0.6396 0.0022 + YFR046C CNN1 0.0036 0.5712 0.0020 - YDR220C 0.0038 0.5919 0.0022 - YPL025C 0.0041 0.6844 0.0028 - YBR006W UGA2 0.0043 0.7684 0.0033 - YBR108W 0.0044 0.4390 0.0019 - YFL034W 0.0046 0.3287 0.0015 - YCL027W FUS1 0.0047 0.7170 0.0034 - YLR308W CDA2 0.0049 0.3424 0.0017 YGR014W MSB2 0.0050 0.6554 0.0033 YJL202C 0.0052 0.6416 0.0033 a The ranking of haploid-specific gene expression determined by analysis of microarray data [11]. b The ranking of potential a1-α2 target of haploid-specific genes. c A + indicates that the a1-α2 binds to the promoter by ChIP assay. Figure 2 ChIP assays of a1-α2 binding to promoter fragments that are not predicted to be targets of the complex. ChIP assays were performed as described in Figure 1. ChIP assays for a1-α2 binding to the FUS1, BUD3, NEM1, CAT2, YFL034W and GUP2 promoters are shown. Comparison with binding site identification by the weight matrix method We compare the performance of our algorithm with that of the weight matrix method [15-18]. In our study, we derived our parameters from a set of artificial sequences. Usually, the weight matrix has to be constructed from a set of known sites. We calculate the weight matrix for a1-α2 from regulatory elements upstream several known target genes: HO, GPA1, FUS3, AXL1, STE5, RME1 and MATα1. As usual, one is faced with a choice of threshold weight matrix score for selecting putative sites in the yeast genome. For a stringent threshold that corresponds to the top 16 targets, we recovered all the genes, other than RME1, used in construction of the weight matrix. However, we did not recover most of the other genuine targets identified, and verified, in this study. If we set the threshold to be lax enough to include RME1, we obtained 55 candidate genes, including STE18 and RDH54, but still miss targets like STE4. It is likely that most of the 55 putative targets are false positives, as evidenced by lack of haploid-specific regulation in the corresponding gene expression data. Overall, we find our method to be more successful than the weight matrix method. The use of mutational data as opposed to literature based data for sequence preference possibly accounts for part of the success (an advantage we may not have for some other transcription factors). However, much of our success has to do with cutting down of false positive rates by using microarray data judiciously. Analysis of all potential a1-α2 target sites in the genome Among the genes identified in the computational analysis, there is a good correlation between the presence of strong a1-α2-binding sites in their promoter region and repression in diploid cells. This raises the question of whether all a1-α2-binding sites function as repressor sites. To address this question we searched for all potential binding sites in the yeast genome. As expected, many of the best sites are in the promoters of known or previously identified haploid-specific genes (Table 1). However, we also identified a number of putative a1-α2-binding sites within ORFs (Table 3). To test if the a1-α2 complex is able to bind to these sites we performed electrophoretic mobility shift assays (EMSAs) with purified α2 and a1 proteins and radiolabeled oligonucleotides containing these sites (Fig 3A). The a1-α2 complex bound to sites from the YKL162C, CDC25, PRM8, PRM9, and URB1 ORFs with weaker affinity than to a strong binding site from the HO promoter, HO(10). However, these sites did have slightly better binding affinity than to the HO(8) site, which we have shown is unable to repress transcription on its own (Mathias and Vershon, unpublished). Table 3 Potential a1-α2 target sites in ORFs ORF Gene Expression p-vala Binding p-valb Combined p-val a1-α2 ChIPc EMSAd YKL014C URB1 0.621 0.042 0.025 - 25× YGL053W PRM8 0.291 0.011 0.002 - 5× YAR031W PRM9 0.891 0.013 0.012 - 10× YKL162C 0.782 0.017 0.014 - 10× YLR310C CDC25 0.102 0.018 0.002 - 5× YPL061W ALD6 0.597 0.018 0.009 - YBR028C 0.793 0.046 0.036 - YOL022C 0.563 0.023 0.013 - YJR016C ILV3 0.248 0.025 0.006 - YBR218C PYC2 0.324 0.027 0.008 - YFR040W SAP155 0.234 0.165 0.038 - YMR269W 0.332 0.049 0.016 - YJL129C TRK1 0.297 0.083 0.024 - YCR053W THR4 0.607 0.073 0.044 - a The ranking of haploid-specific gene expression determined by analysis of microarray data [11]. b The ranking of potential a1-α2 target of haploid-specific genes. c A + indicates that the a1-α2 binds to the promoter by ChIP assay. d Relative binding affinity in fold decrease in affinity compared to the HO(10) binding site. Figure 3 a1-α2 binding in vitro and in vivo to sites in the ORF regions of the genome. (A) An EMSA of purified a1 and α2 proteins binding to a strong a1-α2 binding site, HO(10) (lanes 1–8) and sites from the YKL162C (lanes 9–13), CDC25 (lanes 14–18), a weak a1-α2 binding site from the HO promoter, HO(8) (lanes 19–23), PRM8 (lanes 24–28), PRM9 (lanes 29–33) and URB1 (lanes 34–38). The concentration of the a1 protein was held constant at 1.4 × 10-6 M (lanes 2 and 4–38) and mixed with 5-fold dilutions of the α2 protein starting at 8.2 × 10-8 M (lanes 3, 4, 9, 14, 19, 24, 29 and 34)). The EMSAs shown are phosphorimages of the gels. Lane 1 contains HO(10) probe alone, and lanes 2 and 3 contain 1.4 × 10-6 M a1 and 8.2 × 10-8 M α2 respectively. The fold repression by each site in the context of a heterologous promoter is shown below each gene/promoter. (B) ChIP assays for the genes YKL162C, CDC25, PRM8, PRM9 and URB1 are shown. ChIP assays were performed as described in Figure 1 Since the a1-α2 complex was able to bind to these sites with weak to moderate affinity in vitro, it is possible these sites may partially repress transcription on their own. To test this model, we cloned these sites into the context of the CYC1 promoter driving expression of a lacZ gene and measured the ability of the sites to repress transcription of the reporter in diploid cells [9]. The sites from the CDC25 and URB1 ORFs did not repress transcription of the reporter promoter in diploid cells (Fig 3A). However, the site from PRM8 ORF, which showed the highest binding affinity among the sites found in ORF regions, weakly (2.8-fold) repressed the reporter promoter. This result indicates that this site can function as a repressor site in vivo if placed in the proper context. We next tested whether a1-α2 bound to these sites in the normal genomic context in vivo by ChIP assays. None of the sites in the ORF regions were bound by the a1-α2 complex (Fig 3B and Table 3). This result indicates that while they are competent for weak binding and repression in a heterologous promoter, they are unable to repress transcription in their normal genomic context. Our search also identified several potential a1-α2 binding sites in the promoter regions of genes that do not appear to be repressed in diploid cells (Table 4). Only the COX13 site had moderate binding affinity for the a1-α2 complex in the EMSAs (Fig 4A). However, despite the relatively weak binding affinity of these sites, they were able to partially repress transcription of the reporter in diploid cells (Fig 4A). In particular, the sites from the COX13 and REX2 promoters showed significant levels of repression. Interestingly, although these sites functioned as repressor sites in the context of the heterologous reporter, except for the COX13 promoter, most of these sites were not bound by the a1-α2 complex at their genomic locations by ChIP assays (Fig 4B). These results suggest that the genomic context of most of these a1-α2 sites prevents binding by the complex. Table 4 Potential a1-α2 Target Sites in the Promoters of Non-Haploid-specific Genes ORF Gene Expressiona p-val Binding p-val Combined p-val a1-α2 ChIPb YGL191W COX13 0.4111 0.0053 0.0021 + YPL099C FMP14 0.1622 0.0075 0.0012 YLR059C REX2 0.1532 0.0098 0.0015 +/- YDR212W TCP1 0.5694 0.0017 0.0010 YJL124C LSM1 0.0830 0.0120 0.0010 +/- YAR033W MST28 0.7419 0.0133 0.0099 - YMR015C ERG5 0.6021 0.0138 0.0083 - YMR291W 0.2659 0.0218 0.0058 - YPL188W POS5 0.2648 0.0230 0.0061 +/- YDR101C ARX1 0.3694 0.0298 0.0110 + YHR058C MED6 0.4507 0.0350 0.0157 YGL117W 0.5577 0.0370 0.0206 YIL027C KRE27 0.5762 0.0381 0.0220 a The ranking of haploid-specific gene expression determined by analysis of microarray data [11]. b A + indicates that the a1-α2 binds to the promoter by ChIP assay. A +/- indicates weak (2-fold) enhancement of the band in a/α cells by ChIP assay. Figure 4 a1-α2 binding in vitro and in vivo to putative binding sites in promoters associated with genes which are not expressed in a haploid-specific manner. (A) An EMSA of purified a1 and α2 proteins binding to a strong a1-α2 binding site, HO(6) (lanes 1–7), COX13 (lanes 8–11), REX2 (lanes 12–15), LSM1 (lanes 16–19) and FMP14 (lanes 20–23). The concentration of the a1 protein was held constant at 1.4 × 10-6 M (lanes 2 and 4–23) and mixed with 5-fold dilutions of the α2 protein starting at 8.2 × 10-8 M (lanes 3, 4, 8, 12, 16 and 20). The EMSAs shown are phosphorimages of the gels. Lane 1 contains HO(6) probe alone, and lanes 2 and 3 contain 1.4 × 10-6 M a1 and 8.2 × 10-8 M α2 respectively. The fold repression by each site in the context of a heterologous promoter is shown. (B) ChIP assays for COX13, REX2, LSM1 and FMP14 were performed as described in Figure 1. Discussion Genome-wide gene expression data using SAGE or DNA microarrays has provided a wealth of information on the regulation of genes under certain conditions or by specific transcription factors. The combination of this information with sequence analysis programs has enabled researchers to identify potential regulatory sites. For example, in a pioneering paper, Tavazoie et al. clustered expression data and used multiple local sequence alignment algorithms on the promoter regions of the co-clustered genes to discover regulatory motifs [19]. This approach has been further refined by using Bayesian networks to incorporate additional constraints regarding relative positions and the orientations of the motifs [20]. Another approach has been to break the genes into modules and perform module assignments and motif searches at the same time via an expectation maximization algorithm (as opposed to clustering first and finding motifs later) [21,22]. Although these approaches have worked well at identifying potential targets sites one drawback is that the expression patterns have to cluster well for these methods to work. For a small number of microarray experiments, this may always not be the case. A method that does not utilize clustering is a regression model based analysis to locate "words" in the promoter that correlates with modulation of expression [23]. However, this approach is restricted to retrieving functional consensus binding sites in the promoter regions and for transcription factors with low sequence specificity, this approach needs to be modified. Most of these approaches attack the difficult problem of what to do when relatively little is known about the regulatory system and sequence recognition by the protein. Consequently they develop pattern recognition algorithms that are essentially unsupervised. Our focus has been to take advantage, as much as possible, of knowledge about the biological system and use that information combined with expression analysis to identify potential target sites. The minor loss of generality of the tools resulting from such an approach is more than offset by its predictive power. To determine if the changes in expression of a specific gene are the result of a transcription factor working at the promoter we developed an algorithm that combines expression data with information on the binding site preference for a transcription factor. As a test for this algorithm we identified genes in yeast that are direct targets for regulation by the a1-α2 repressor complex. We also used this method to identify genes that are repressed in diploid cells but that are not direct targets of the complex, as well as functional a1-α2 binding sites that do not appear to repress transcription in their genomic context. The combination of these sets of findings has provided insight into the regulatory network and mechanism of repression by the a1-α2 complex. The primary goal of this study was to identify genes that are direct targets for repression by the a1-α2 complex. There are two major functional subsets among the a1-α2 target genes identified in this analysis (Table 1). One, not surprisingly, involves genes that are required for various processes in mating of the two haploid cell-types. These include components of the mating pheromone signal transduction pathway, such as GPA1, STE18, STE4, and STE5, which are activated in response to the binding of pheromone from the other cell type [24]. This group also includes genes further down that pathway, such as FAR1 and FUS3, which are required for cell-cycle arrest before mating. A number of these genes have previously been shown or suspected to be under the control of a1-α2 repressor complex [25,26]. Repression of these genes in diploid cells is biologically important because it prevents further mating by diploid cells. If diploid cells mate they would form triploids or higher ordered genomic polyploids, which are genetically unstable during meiosis and therefore detrimental to cell survival. The second subset of genes identified in the analysis is associated with mating type switching and recombination. The HO gene is a known target of the a1-α2 complex and its promoter contains 10 binding sites of varying affinity [25]. Repression of HO is essential in diploid cells because it prevents switching of one of the MAT loci to form homozygous a/a or α/α diploid cells. Although diploid in genomic content, cells homozygous for the MAT loci are competent to mate and therefore would form higher order genomic polyploids that are genetically unstable. We have also shown that NEJ1, which is involved in non-homologous end-joining (NHEJ), is a direct target for the a1-α2 complex [27,28]. It has been proposed that that repression of the NHEJ pathway may promote homologous recombination and crossing over in diploid cells. In addition, we found that RDH54, a gene involved in double-stranded DNA break repair, is a direct target for the a1-α2 complex [29]. This result is somewhat unexpected because RDH54 is required for meiosis and null mutants show significantly reduced spore viability. It is likely that the a1-α2 complex only partially reduces the level of expression of the gene and that diploid cells require a lower level of activity of the protein. We also identified several genes that fell outside of these two subsets. One is RME1, which encodes a transcriptional repressor of IME1, the master regulator of meiosis [30-32]. a1-α2-mediated repression of RME1 is required to allow cells to enter the meiotic pathway in diploid cells. Interestingly, we also found that PDE1 and MET31 are weakly, but reproducibly, direct targets for repression by the a1-α2 complex. The Pde1 protein is a low affinity cAMP phosphodiesterase that appears to have a role in response to stress and cell aging [33]. Repression of PDE1 in diploids may partially account for the difference of starvation response between haploids and diploids. Met31 is a zinc finger DNA-binding protein that activates genes involved in sulfur metabolism [34]. It is unclear why this gene would be a target for the a1-α2 complex. It is possible that the presence of an a1-α2 target site upstream of a gene that has lower expression in diploid cells was fortuitous and that these sites were not functional targets. However, if this was the case then there would be little pressure to conserve these binding sites through evolution. Several closely related species of yeast have been sequenced and comparison of the corresponding promoter regions has led to the discovery of conserved regulatory motifs [35,36]. Although lack of conservation does not imply non-functionality, significant conservation strongly argues for functionality of a putative regulatory element. To investigate this possibility, we performed a phylogenetic comparison to infer whether these sites are preserved among six sequenced Saccharomyces species using the PhyloGibbs program [37]. The program identified the a1-α2 binding site among a promoter set including many known haploid-specific genes (HO, NEJ1, GPA1, STE4, and STE18). This analysis also showed that the a1-α2 binding sites in the RDH54, PDE1and MET31 promoters are strongly conserved among multiple species, suggesting that these sites play an important functional role. Our analysis identified a number of haploid-specific genes that do not appear to be direct targets of the a1-α2 repressor complex (Table 2). Genes in this list do not contain a recognizable a1-α2-binding site and, with the exception of NEM1, are not detectably bound by the a1-α2 complex in the ChIP assays. It is possible that a1-α2 indirectly turns off these genes by repressing an activator protein that is required for their expression. However, besides MET31, there were no obvious genes coding for activator proteins that were direct targets of the a1-α2 complex. It is possible that the haploid-specific genes without a1-α2 sites are indirectly repressed through more complex mechanisms that involve repression of RME1. We also identified potential a1-α2-binding sites in the genome that do not appear to repress expression of nearby genes. Although sites from the PRM8, PRM9, CDC25, and LSM1 promoters appear to be moderate binding sites for the a1-α2 complex in vitro, ChIP and heterologous reporter assays showed these sites are neither bound by the proteins nor are functional repressor sites in vivo. Many of these sites lie in open reading frames of actively transcribed genes and so it is possible that transcription through the binding site or the chromatin structure of the region prevents high affinity binding by the complex. The model that the genomic context of these sites is important for their regulatory activity is further supported by our results that show that some of these sites, such as COX13 and REX2, function as strong a1-α2 dependent repressor sites in the context of the heterologous promoter. Although a1-α2 complex is bound to the COX13 site in vivo it does not appear to repress transcription of this gene in diploid cell. Interestingly, this binding site is very close to the end of the coding region of IME4, an inducer of meiosis that is expressed in diploid cells [38]. The IME4 gene is only expressed in diploid cells and it was thought that the a1-α2 complex may be indirectly activating its expression by repressing a repressor protein, such as RME1. However, the fact that a1-α2 binds to the downstream region of this gene suggests that it may play a direct role in its expression. Our data shows that the algorithm we have developed is useful in sorting between direct and indirect targets of a transcription factor. Although we have used mutational data to define the binding site for the a1-α2 complex, in principal binding site sequences derived from site selection experiments may also be used. This analysis may also complement genome-wide ChIP studies to identify the target sites of the transcription factor. Conclusions In summary, we show that combining microarray data with motif analysis, lets us distinguish between the genes that are direct targets of a transcription factor and those that are modulated because of secondary effects. We get excellent agreement of the computational predictions with location analysis by ChIP experiments. We find most of the direct targets of a1/α2 complex to be involved in the mating pathway, mating type switching, recombination and meiosis. We also found a few weak targets that are possibly involved in sensing and control of the metabolic state. We also see that the sites we predict solely based on single species data are often evolutionarily conserved in other species of Saccharomyces. Methods Combined scoring of genes from microarray data and mutational analysis We define a scoring algorithm that ranks gene expression patterns. For gene g, the score is given in terms of the expression in different types of cells (a, α and a/α) Score(g) = sgn((Xa(g) + Xα(g))/2 - Xa/α(g)) [(Xa(g)) + Xα(g))/2 - Xa/α(g)]2 - A(Xa(g) - Xα(g))2. We initially used the logarithm of expression level of the gene g for the three cell types for the variables Xt(g) with t indicating the type. We have since found that using the complete expression data for the polyploids is a better strategy. In the final results, shown in the paper, Xa(g) is the average of the log expression for a, aa and aaa. Likewise, Xα (g) is the average from α, αα and ααα. Xa/α(g) comes from averaging log expression over aaα, aαα and aaαα. The polyploid averaged quantities tend to be less noisy (demonstrated, for example, by the quantities Xa and Xα being close to each other for the generic gene, which is not regulated by cell type. This, in turn, allows easier detection of genuine haploid-specific targets. An explanation of the purpose served by different terms in the overall score is described below. The first term scores well when expression in diploids is lower than the average expression in haploids. The second term penalizes the gene if the expressions in different types of haploids are very different. A is chosen to be large enough so that known a-specific genes and α-specific genes score worse than known haploid-specific genes, but not large enough to overwhelm the first term. The optimal A is about 10. Comparison of the performance of our algorithm for A = 1 and A = 10, shows that the biologically known sites almost always stay near the top but further down in the list the second choice is better. The exception is a special gene: MATα1. Since MATα1 is not present in the MATa type cell, there is a penalty for expression patterns. Cumulative probability for any gene to have higher score than a gene g is Pexprn(g), namely, fraction of genes with score higher than g. This scoring function would rank haploid-specific genes high but may not select out genes that are directly regulated by the a1-α2 repressor complex. In order to select for genes with an upstream region with a strong a1-α2 repressor, we used the binding site mutational data available [9]. In these experiments, repression of a heterologous promoter, incorporating single site mutations of a consensus binding site of the a1-α2 repressor, was measured. Under the assumption that the degree of repression is inversely proportional to how often that site is occupied, we derived the expression: 1/Repression ∝ [1 - 1/(1 + eβE(S)/z)] ≈ eβE(S)/z assuming near saturation of binding. The symbol z represents the fugacity and β is inverse of kBT, kB being the boltzman constant. The binding (free) energy is given by E(S) = ΣibεibSib, within the single base model [15,16]. The index i runs over the positions in the motif and b runs over the bases A, C, G, T.Sib is 1 or 0 depending upon whether the i-th base is b or not. The parameters εib represent effects of single base changes on the binding (free) energy. They are related to the weight matrix parameters [17,18] widely used to characterized variable motifs. Note that eβE(S)/z would more commonly be represented as (K/ [Protein])•exp(ΔG(S)/RT) in the biochemistry literature [18,39]. The independent base model is only an approximation and mutations in nearest neighbor sites could produce effects that we cannot estimate from the existing data. There is a better separation between well-known sites and generic sequences if an extra penalty is added to the score for neighboring base pairs which both differ from the consensus. In this way every base different from consensus and neighboring another base different from consensus draws an additional penalty to the binding energy score. This parameter was set to be ln(2), by experience. Although this method prevented many false positives, it also penalized a few genuine candidates, such as the binding sites in the promoters of FAR1 and MATα1. Thus, from the effect of the single base mutations, we estimated the parameters εib. Armed with these parameters, we found the probability P(E|ε, L) that a random sequence of a certain length, L, would have a subsequence of binding energy greater than E. For gene g, the strongest site in the upstream region of length L would have binding energy Eg. Low values of Pbinding(g) = P(Eg|ε,L), indicated the presence of a good binding site. The genes were ordered according to the lowest value of a combined p-value, Pexprn(g) Pbinding(g), and then ranked as candidates for haploid-specific genes that are directly repressed by the a1-α2 complex. One of the issues in such studies is how to decide on how many of the top candidates are significant. This problem occurs even in solely, sequence based analysis as well [40]. In our study, we generated random combinations of expression p-values with scrambled binding p-values, so that we could choose a cutoff threshold by comparing the ordered p-values with the ordered "scrambled" p-values. Figure 5 plots these sorted p-values against average (log) sorted p-values for the random combinations. The comparison suggests that only about 10–15 top candidates in the list are significant. Weight matrix based search A weight matrix [15-18] search for binding sites was performed using a set of known sites [9] to construct the matrix. Each matrix entry, wia, was set to log((fia + δ)/(Pa + δ)), where fia is the frequency with which base a appears in the ith position in the known sites, Pa is the frequency with which base a appears in the promoters of genes and δ is a small number added to ensure that the weight matrix score is finite even when fia = 0. Each subsequence, Sia, of length 20 in each promoter was assigned a weight matrix score Σia Sia wia. After a threshold score is chosen, sites scoring above that threshold are declared to be binding sites. Automated primer generation An automated procedure for generating primers flanking a specified site in the genome sequence, σ, was implemented. To each pair of numbers, du, and dd, representing primer distances upstream and downstream of the candidate binding site respectively, and primer lengths lu, and ld, a score is assigned via S(du, dd, lu, ld,σ) = - Σaka(Pa(du, dd, lu, ld,σ) - )2 where the Pa are functions including the melting temperatures, distances of upstream and downstream primers from the candidate site, total length of the bound region, and the difference between the primer melting temperatures. The s are the preferred values of those functions. The ka are adjusted to reflect the relative importance of the parameters; for example it is more important that the difference in melting temperatures be close to zero than it is that the distance to the upstream primer match the distance to the downstream primer. Values of du, dd, lu and ld are restricted to those whose corresponding primers have GG, GC, CG, or CC at the end nearest the candidate site. Primers are identified by selecting the values of du, dd, lu and ld which maximize S. [A web-based interface to this algorithm is available at ] Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was carried out as described previously [41] with the following modifications. One liter of JRY103 (MATα/MATaade2-1/ADE2 HIS3/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1 ash1Δ::LEU2/ash1Δ::LEU2) and JRY118 (MATα/mataΔ::TRP1 ade2-1/ADE2 HIS3/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1 ash1Δ::LEU2/ash1Δ::LEU2) cultures were grown to an A600 of 0.5 and treated with 1% formaldehyde for 20 min at RT on a rotating shaker at low speed. Cells were collected, washed 2X with cold 1XTBS. Equal volumes of cells were aliquoted into ten 1.5 ml microfuge tubes, washed once with 1.5 ml of cold 1X TBS. The pellets in each tube were resuspended with 400 μl of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) plus 1 mM PMSF, 1 mM benzamidine, and 1X Protease inhibitor cocktail from Roche (Cat No. 1873580) and also manufacturer recommended concentration of protease inhibitor cocktail from SIGMA (Cat No., P 8215). To this 200 μl of glass beads were added to each tube and lysed using a multitube vortexer at full speed for 30 min at 4°C. The lysate was transferred in a new tube and 400 μl of lysis buffer was added and vortexed briefly. The lysates were centrifuged at 12,000 g for 10 min at 4°C and the supernatants were sonicated at 30% output for four 10 sec cycles with intermittent cooling on ice. The lysates were cleared by centrifugation at 12,000 g for 10 min and 1 mM PMSF was added to the samples. A 1/10th volume aliquot was removed and frozen to be used as total chromatin control. The remaining sample was precleared by the addition of 25 μl recombinant protein G-agarose beads, incubated while nutating for 30 min and the supernatant was collected after centrifugation at 12,000 for 5 min. 1 μl of rabbit anti-α2 antiserum (a gift from A. Johnson, UCSF) was added to each supernatant of the samples and incubated 12 h on a nutator at 4°C. To immunopreciptiate α2 50 μl of recombinant protein G agarose beads (Roche) was added to the samples and nutated for 90 minutes at 4°C. The protein G beads were pelleted, washed once in low salt buffer (0.1%SDS, 1% Triton X-100, 20 mM Tris pH8.0, 2 mM EDTA and 150 mM NaCl), once in high salt (composition same as lowsalt + 500 mM NaCl), once in LiCl buffer (0.25 M LiCl, 1% IGEPAL, 1XTE and 1% Na-Deoxycholate) and twice with 1XTE (pH8.0). The immunoprecipitated DNA was eluted twice with 250 μl of elution buffer (1%SDS and 0.1 M NaHCO3) and the eluates were pooled (500 μl final volume). To this 20 μl of 5 M NaCl was added and incubated 12 h at 65°C. To remove the crosslinks, 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl, pH 7.5 and 2 μl of proteinase K (10 mg/ml) was added and incubated for 45 minutes at 45°C. The DNA samples were extracted once with Phenol:chloroform:Isoamylalcohol and the DNA was ethanol precipitated, washed once with 70% ethanol and resuspended in 50 μl (IP) or 500 μl (TC) TE. Purified DNA from the immunoprecipitated samples was subjected to multiplex PCR amplification with primers specific for the STE6 promoter as a positive control for the immunoprecipitation of α2 and the YDL223C ORF as a negative control for nonspecific immunoprecipitation, along with the specific primers for candidate α2-a1 target sites. PCRs were carried out in 50 μl containing 10 pmols of each primer, 0.2 mM dNTPs, 2 mM MgCl2, 1X Eppendorf Taq buffer, 0.5X Taq Master buffer and 2.5 U of Eppendorf Taq polymerase. The amplifications were carried out at 94°C for 1 min and 30 secs, followed by 25 cycles of 94°C for 30 secs, 52°C for 1 min, and 72°C for 30 secs and a final extension step of 7 min at 72°C. The PCR products were separated on 2.5% agarose gels. Electrophoretic mobility shift assays Oligonucleotides containing the predicted a1-α2 binding sites from within the ORFs of URB1, PRM8, PRM9, YKL162C and CDC25 and the promoters of COX13, REX2, LSM1, and FMP14 were synthesized, one strand was end-labeled with [γ-32P]-ATP, and then annealed with excess cold complementary oligonucleotide. The HO(10) and HO(8) a1-α2 sites within Upstream Regulatory Sequence 1 (URS1) of the HO promoter were used as strong and weak binding sites respectively. The EMSA was performed as described previously [42], using a constant 1.4 μM a1 and five-fold titrations of α2 starting at 82 nM in protein dilution buffer (50 mM Tris pH 7.6. 1 mM EDTA, 500 mM NaCl, 10 mM 2-mercaptoethanol, 10 mg/ml bovine serum albumin). β-galactosidase assays Oligonucleotides containing a1-α2 binding sites were synthesized with 5' overhangs to allow cloning into the XhoI site of pTBA23 (2μ URA3 Ampr), a reporter plasmid containing a CYC1-lacZ fusion [43]. Reporter constructs were transformed into JRY103 and JRY118 and the β-galactosidase activity was measured on three independent transformants, as described previously [14]. List of Abbreviations SAGE – Serial Analysis of Gene Expression HD – Homeodomain ORF – Open Reading Frame ChIP – Chromatin immunoprecipitation NHEJ – Non-Homologous End Joining TC – Total Chromatin IP – Immunoprecipitated sample EMSA – Electrophoretic Mobility Shift Assay PCR – Polymerase Chain Reaction PMSF – Phenyl Methyl Sulfonyl Fluoride EDTA – Ethylenediaminetetraacetic Acid Author's Contribution VHN carried out the ChIP experiments, and wrote parts of the first draft of the manuscript. RAO did the bioinformatics analysis, and wrote programs facilitating primer design. ARB did EMSA and beta-gal assays. JRM constructed the strains and contributed to the design of the ChIP experiment. AKV and AMS supervised and coordinated the computational and the experimental research as well as prepared the manuscript. All authors contributed to the manuscript and approved the final version. Figure 5 Significance of combined p-values. Natural logarithm of combined p-values for twenty permutations/scrambles generated, sorted and plotted (in blue) against average of log p-value. The genuine combined value is plotted in red. Acknowledgements We thank Alexander Johnson for the rabbit α2 antibody and Yvette Green for help with some of the initial ChIP assays. We also thank Rahul Siddharthan for running PhyloGibbs on the candidate direct target promoter set. JM was supported by a Charles and Johanna Busch predoctoral fellowship. This work was partially supported by a grant from the National Institutes of Health to AKV (GM49265). ==== Refs Lockhart DJ Dong H Byrne MC Follettie MT Gallo MV Chee MS Mittmann M Wang C Kobayashi M Horton H Brown EL Expression monitoring by hybridization to high-density oligonucleotide arrays Nat Biotechnol 1996 14 1675 80 9634850 10.1038/nbt1296-1675 DeRisi JL Iyer VR Brown PO Exploring the metabolic and genetic control of gene expression on a genomic scale Science 1997 278 680 6 9381177 10.1126/science.278.5338.680 Velculescu VE Vogelstein B Kinzler KW Analysing uncharted transcriptomes with SAGE Trends Genet 2000 16 423 5 11050322 10.1016/S0168-9525(00)02114-4 Johnson A McKnight SL, Yamamoto KR A combinatorial regulatory circuit in budding yeast In Transcriptional Regulation 1992 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 975 1006 Goutte C Johnson AD Recognition of a DNA operator by a dimer composed of two different homeodomain proteins EMBO J 1994 13 1434 42 7907979 Li T Stark MR Johnson AD Wolberger C Crystal structure of the MATa1/MATα2 homeodomain heterodimer bound to DNA Science 1995 270 262 269 7569974 Wolberger C Vershon AK Liu B Johnson AD Pabo CO Crystal structure of a MATα2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions Cell 1991 67 517 528 1682054 10.1016/0092-8674(91)90526-5 Zhong H Vershon AK The yeast homeodomain protein MATalpha2 shows extended DNA binding specificity in complex with Mcm1 J Biol Chem 1997 272 8402 9 9079665 10.1074/jbc.272.14.9464 Jin Y Zhong H Vershon AK The yeast a1 and alpha2 homeodomain proteins do not contribute equally to heterodimeric DNA binding Mol Cell Biol 1999 19 585 93 9858582 Mathias JR Zhong H Jin Y Vershon AK Altering the DNA-binding specificity of the yeast matalpha 2 homeodomain protein J Biol Chem 2001 276 32696 703 11438530 10.1074/jbc.M103097200 Galitski T Saldanha AJ Styles CA Lander ES Fink GR Ploidy regulation of gene expression Science 1999 285 251 4 10398601 10.1126/science.285.5425.251 Zhong H McCord R Vershon AK Identification of target sites of the alpha2-Mcm1 repressor complex in the yeast genome Genome Res 1999 9 1040 7 10568744 10.1101/gr.9.11.1040 Passmore S Maine GT Elble R Christ C Tye BK Saccharomyces cerevisiae protein involved in plasmid maintenance is necessary for mating of MAT alpha cells J Mol Biol 1988 204 593 606 3066908 Keleher CA Passmore S Johnson AD Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1 Mol Cell Biol 1989 9 5228 30 2689875 Berg OG von Hippel PH Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters J Mol Biol 1987 193 723 50 3612791 Berg OG von Hippel PH Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites J Mol Biol 1988 200 709 23 3045325 Staden R Computer methods to locate signals in nucleic acid sequences Nucleic Acids Res 1984 12 505 19 6364039 Stormo GD Fields DS Specificity, free energy and information content in protein-DNA interactions Trends Biochem Sci 1998 23 109 13 9581503 10.1016/S0968-0004(98)01187-6 Tavazoie S Hughes JD Campbell MJ Cho RJ Church GM Systematic determination of genetic network architecture Nat Genet 1999 22 281 5 10391217 10.1038/10343 Beer MA Tavazoie S Predicting gene expression from sequence Cell 2004 117 185 98 15084257 10.1016/S0092-8674(04)00304-6 Segal E Shapira M Regev A Pe'er D Botstein D Koller D Friedman N Module networks: identifying regulatory modules and their condition-specific regulators from gene expression data Nat Genet 2003 34 166 76 12740579 10.1038/ng1165 Segal E Yelensky R Koller D Genome-wide discovery of transcriptional modules from DNA sequence and gene expression Bioinformatics 2003 19 i273 82 12855470 10.1093/bioinformatics/btg1038 Bussemaker HJ Li H Siggia ED Regulatory element detection using correlation with expression Nat Genet 2001 27 167 71 11175784 10.1038/84792 Sprague GF Thorner JW Broach JR Pheromone response and signal transduction during mating process of Saccharomyces cerevisiae In The Molecular and Cellular Biology of the Yeast Saccharomyces – Gene Expression 1992 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 657 744 Miller AM MacKay VL Nasmyth KA Identification and comparison of two sequence elements that confer cell-type specific transcription in yeast Nature 1985 314 598 603 3887184 Liu H Styles CA Fink GR Elements of the yeast pheromone response pathway required for filamentous growth of diploids Science 1993 262 1741 4 8259520 Valencia M Bentele M Vaze MB Herrmann G Kraus E Lee SE Schar P Haber JE NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae Nature 2001 414 666 9 11740566 10.1038/414666a Kegel A Sjostrand JO Astrom SU Nej1p, a cell type-specific regulator of nonhomologous end joining in yeast Curr Biol 2001 11 1611 7 11676923 10.1016/S0960-9822(01)00488-2 Klein HL Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae Genetics 2001 157 557 65 11156978 Mitchell AP Herskowitz I Activation of meiosis and sporulation by repression of the RME1 product in yeast Nature 1986 319 738 42 3513021 Kassir Y Granot D Simchen G IME1, a positive regulator gene of meiosis in S. cerevisiae Cell 1988 52 853 62 3280136 10.1016/0092-8674(88)90427-8 Covitz PA Herskowitz I Mitchell AP The yeast RME1 gene encodes a putative zinc finger protein that is directly repressed by a1-alpha 2 Genes Dev 1991 5 1982 9 1936989 Ma P Wera S Van Dijck P Thevelein JM The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling Mol Biol Cell 1999 10 91 104 9880329 Blaiseau PL Isnard AD Surdin-Kerjan Y Thomas D Met31p and Met32p, two related zinc finger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism Mol Cell Biol 1997 17 3640 8 9199298 Cliften P Sudarsanam P Desikan A Fulton L Fulton B Majors J Waterston R Cohen BA Johnston M Finding functional features in Saccharomyces genomes by phylogenetic footprinting Science 2003 301 71 6 12775844 10.1126/science.1084337 Kellis M Patterson N Endrizzi M Birren B Lander ES Sequencing and comparison of yeast species to identify genes and regulatory elements Nature 2003 423 241 54 12748633 10.1038/nature01644 Siddharthan R van Nimwegen E Siggia ED PhyloGibbs: A Gibbs sampler incorporating phylogenetic information In The First Annual RECOMB Satellite Workshop on Regulatory Genomics 2004 University of California, San Diego Shah JC Clancy MJ IME4, a gene that mediates MAT and nutritional control of meiosis in Saccharomyces cerevisiae Mol Cell Biol 1992 12 1078 86 1545790 Liu X Clarke ND Rationalization of gene regulation by a eukaryotic transcription factor: calculation of regulatory region occupancy from predicted binding affinities J Mol Biol 2002 323 1 8 12368093 10.1016/S0022-2836(02)00894-X Djordjevic M Sengupta AM Shraiman BI A biophysical approach to transcription factor binding site discovery Genome Res 2003 13 2381 90 14597652 10.1101/gr.1271603 Kuo MH Allis CD In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment Methods 1999 19 425 33 10579938 10.1006/meth.1999.0879 Hart B Mathias JR Ott D McNaughton L Anderson JS Vershon AK Baxter SM Engineered improvements in DNA-binding function of the MATa1 homeodomain reveal structural changes involved in combinatorial control J Mol Biol 2002 316 247 56 11851335 10.1006/jmbi.2001.5333 Acton TB Zhong H Vershon AK DNA-binding specificity of Mcm1: operator mutations that alter DNA-bending and transcriptional activities by a MADS box protein Mol Cell Biol 1997 17 1881 9 9121436

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==== Front BMC ImmunolBMC Immunology1471-2172BioMed Central London 1471-2172-5-191534503010.1186/1471-2172-5-19Research ArticleExonuclease activity and P nucleotide addition in the generation of the expressed immunoglobulin repertoire Jackson Katherine JL [email protected] Bruno [email protected] William [email protected] Andrew M [email protected] School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia2 Garvan Institute of Medical Research, Sydney, Australia3 St Vincent's Clinical School, University of New South Wales, Sydney, Australia2004 2 9 2004 5 19 19 21 5 2004 2 9 2004 Copyright © 2004 Jackson et al; licensee BioMed Central Ltd.2004Jackson et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. Results A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3' D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition. Conclusions The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the 'palindromic' nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences. ==== Body Background The variable domain of the immunoglobulin heavy chain (IGH) is encoded by the IGHV (variable), the IGHD (diversity) and the IGHJ (joining) genes. In developing B cells these genes are brought together via a process of recombination involving the selection of one of each gene type from sets of genes present within the genome [1]. The bringing together of the selected IGHV, IGHD and IGHJ genes generates combinatorial diversity [1]. The first genes to join are the IGHD and IGHJ genes, followed by the bringing together of the IGHV gene with D-J. Further junctional diversity is generated at the points between the joining genes [2,3]. Junctional diversity results from the loss of nucleotides through the action of unknown exonuclease(s) and from the addition of N [3] and P nucleotides [2]. The final IGH V-D-J rearrangement in mature B cells is finally subject to the process of somatic hypermutation in secondary lymphoid organs which involves the targeted introduction and accumulation of point mutations [4]. The addition of N nucleotides is performed by the enzyme terminal dideoxynucleotidyl transferase (TdT), and in the IGH locus this addition can occur at both the D to J and the V to D-J joins [5]. The regions of N addition are denoted as N regions, and nucleotides that fall between the V and D genes are denoted as N1 regions, while those that lie between the D and J genes are denoted as N2 regions. P nucleotides are derived from the asymmetric opening of hairpin loops that form at gene ends as part of the rearrangement process [6]. The opening of the hairpin loops produces short, self-complementary single stranded extensions that can be incorporated into junctions, or may alternatively be removed via exonuclease activity [6]. It is the self-complementarity of P nucleotides that leads to their palindromic appearance and thus to their name. Hairpin opening is said to produce inserts of 0–4 nucleotides [2]. P nucleotides have been associated with the IGHV and IGHJ genes, as well as with each end of the IGHD gene [7] and estimates of the frequency of P nucleotide addition suggest a presence in about ten percent of sequences [7-10]. The mechanism of immunoglobulin gene rearrangement was first proposed by Tonegawa in the late 1970's [1]. Since that time, much has been learnt about the processes involved. Some areas, however, remain relatively uninvestigated, including the nature of exonuclease removal and the contribution of P nucleotide addition to junctional diversity. The lack of research in these fields may reflect the inherent difficulties in studying the relevant gene sequences, because IGH V-D-J junctions are the result of random and imprecise processes. It can therefore be difficult to distinguish between gene ends and N or P additions. The very few reports of exonuclease removal in the literature mainly describe analysis of murine sequences [11-14]. These investigations revealed nucleotide loss to be significantly different for murine IGHJ and IGHD genes. Differences were seen in the average exonuclease removal from IGHJ and IGHD gene subgroups, with individual gene subgroups possessing significantly different average levels of nucleotide removal. Influences upon gene processing that have been proposed to explain these observations include the presence of TG motifs [15], the relative location of stretches of 3 or more W (A or T) nucleotides and their positional relationship with respect to 2 or more S (G or C) nucleotides [12], and the presence of TAT motifs [13]. Recent advances in data standardisation in immunogenetics has allowed for improved statistical analysis. The standardisation emanates from IMGT-ONTOLOGY [16,17] upon which one of the most widely used immunogenetics tools, IMGT/V-QUEST, is based [18]. IMGT, the IMmunoGeneTics Information SystemR, also offers standardised nomenclature [19] and standardised numbering of positions within immunoglobulin sequences [20]. Despite the importance of IMGT within the field, the tools offered still suffer from shortcomings especially in the analysis of IGH V-D-J junctions. Alternative means of analysing junctions are therefore still sought by researchers [21,22]. The development of a statistically based algorithm for the partitioning of immunoglobulin sequences [21] as an alternative to IMGT/Junction Analysis [18], combined with the large amount of sequence data available through public nucleotide databases, has allowed us to investigate the nature of nucleotide removal from human immunoglobulin heavy chain genes in the expressed repertoire. Improved means of identification of gene ends facilitates the development of datasets with more certain partitioning. This study reports the extent of P nucleotide addition and the nature of exonuclease removal in the expressed human repertoire. Analysis of nucleotide loss and addition within the dataset reveals that different gene subgroups undergo distinct processing by exonuclease(s) and shows that there is no significant contribution by P nucleotides to the diversity of the expressed repertoire. Results Dataset creation The collection of human IgM sequences from public databases resulted in a dataset of approximately 1500 sequences. The exclusion of fetal, moderately and highly mutated (>5 mutations) and disease associated sequences reduced the dataset to 306 sequences. Further exclusions were made of those sequences that showed signs of IGHV gene replacement or the utilization of multiple D genes. Five sequences utilized two D gene segments, as identified using strict criteria as previously described [21] (EMBL:U97246, L12190, L29154, AJ519292, AJ245025). Evidence of IGHV gene replacement, in the form of V gene 'footprints', was seen in 7 sequences (EMBL:L29154, AJ245008, AJ245280, AJ519296, AY003831, X54445 Kabat:AL311). The footprints were unique sequences of 6 or more nucleotides derived from V gene ends containing a cryptic recombination signal sequence (cRSS) which is thought to be essential for replacement events [23-26]. The final dataset contained 294 sequences. Within the final dataset of 294 sequences there were 245 sequences for which IGHV, IGHD, IGHJ, N1 and N2 regions could be defined. A further 49 sequences, lacking determinable D, N1 and N2 region but possessing identifiable IGHV and IGHJ genes were also included in the dataset. For these 49 sequences, it was not possible to confidently determine the utilized IGHD gene within the junctional nucleotides, however, the ends of the V and J regions could be determined accurately. All IGHV, IGHD and IGHJ gene subgroups were represented within the dataset. Details of the dataset can be seen in the Appendix [see Additional File 1]. Exonuclease removals from genes and gene subgroups Exonuclease removal was evident in each of the 245 IGH V-D-J rearrangements examined, with 25% of IGH V-D-J rearrangements displaying removal from all four gene ends. A further 48% of IGH V-D-J sequences had removals from three of the four gene ends. The average number of nucleotides lost from each of the gene ends is presented in Figure 1. Figure 1 Average exonuclease removal from IGH genes. The average nucleotide removal from of the gene ends was examined for 294 IGHV and IGHJ genes and 245 IGHD genes. For the IGHD genes, removals were considered from each end of the gene; 5' (V-D side) and 3' (D-J side). Bars represent standard error. Examination of the 294 IGHV and IGHJ segments revealed 41% of IGHV ends lacked removals, compared to just 18% of IGHJ ends. Sixteen percent of the 245 IGHD genes lacked removals from the 5' end of the D gene, and 17% had no removals from the 3' end of the D region. Exonuclease removals ranged from 0 to 13 nucleotides at the 3' V-REGION and 0 to 14 nucleotides at the 5' D-REGION end. At the D-J junction, 3' D-REGION and 5' J-REGION removals both ranged from 0 to 20 nucleotides. A significant difference in the extent of exonuclease removals was observed between the IGHV, IGHD and IGHJ gene ends (p < 0.0001, Kruskal-Wallis Test). Average removals from IGHV region ends were significantly lower than removals from IGHD and IGHJ region ends (p < 0.001, Dunn's Multiple Comparison Test). On average, only 1.2 nucleotides were lost from IGHV region ends while average removals of 4.7 nucleotides were evident from each of the IGHD region ends as well as from the IGHJ region ends. The average number of nucleotides removed from each gene subgroup within the three genes was calculated to identify differences in processing of sequences at the gene subgroup level (Figure 2). It was necessary to exclude IGHV7 from the analysis as only a single sequence from this subgroup was in the dataset. Average removals differed significantly among the six IGHV subgroups analyzed (p = 0.03, Kruskal-Wallis Test) (Figure 2A). Comparison testing was, however, unable to identify the source of the difference. Figure 2 Average exonuclease removal from gene subgroups. The average exonuclease removals from gene ends was investigated for each IGHV, IGHD and IGHJ subgroup. Significant differences were seen among the 294 IGHV genes (A). No significant difference between the 245 IGHD genes were seen at the 5' end (B). The 3' IGHD end does show significant differences for the IGHD subgroups (C) as do the six IGHJ subgroups (D). Bars represent standard error. Removals from D gene subgroups were examined at each of the region ends. The removals from the 5' D end did not reveal significant differences, but significant differences were seen between the subgroups at the 3' end (p < 0.0001, one-way ANOVA) (Figure 2B, Figure 2C). More extensive removals, of 6.0 and 7.5 nucleotides respectively, were observed from IGHD2 and IGHD3 subgroup members (p < 0.0001, Tukey's Multiple Comparison Test). The remaining 5 IGHD subgroups experienced average deletions of 2.1 nucleotides at their 3' ends. Comparison of average nucleotide loss for each of the six IGHJ subgroups revealed significant differences between the average removals (p < 0.0001, one-way ANOVA). The low level of removals from IGHJ3 was notable. On average just 1.3 nucleotides were removed from IGHJ3 sequences, while an average of 6.4 nucleotides were removed from IGHJ6 sequences (p < 0.002, Tukey's Multiple Comparison Test) (Figure 2D). Influence of W and S motifs A more detailed examination of exonuclease removals from the IGHJ genes was undertaken, to investigate the influence of W and S motifs. The presence of these motifs in the first 15 5' nucleotides of the IGHJ ends was considered. IGHJ ends containing 5' S motifs showed significantly lower average removals than those lacking a 5' S motif (p < 0.0001, Kruskal-Wallis Test). The IGHJ genes whose sequences did not possess an S motif within the first 15 5' nucleotides had, on average, three more nucleotides removed (Figure 3). Figure 3 Influence of W and S motifs on nucleotide loss. IGHJ genes were grouped by the presence of W and S motifs within the first 15 nucleotides of the IGHJ subgroup sequence. Average exonuclease loss was examined for the three sets; 5' W only, 5' S only and S then W. Contribution of P nucleotides to diversity Putative P nucleotides were identified among those gene ends that remained untrimmed by exonuclease activity during the process of IGH V-D-J rearrangement. Examples of nucleotides that satisfied the P nucleotide criteria were observed at 3' V, 5' D, 3' D and 5' J gene ends. The identified nucleotides ranged in length from 1 to 4 nucleotides. The observed P nucleotides fell into twenty-three sets based upon unique sequences, and the gene end at which they were observed (Table 1). Each of the 23 sets was analyzed to determine the likelihood that apparent P nucleotides were actually the result of N additions. The p-values for each of the P nucleotide sequences are shown in Table 1. Correction of the significance level for the comparison of the 23 sets using the Bonferroni adjustment resulted in a required alpha value of 0.003. Table 1 Putative P Nucleotides in a dataset of 294 human IgM sequences Gene Segment Putative P Sequence Observed Total Junctions p-value1 IGHV C 10 111 0.99 T 25 111 0.023 CC 2 105 0.99 TC 10 105 0.049 TG 2 105 0.98 TCT 3 100 0.045 TGT 1 100 0.55 TCTC 1 86 0.21 IGHD 5' A 1 37 0.99 C 5 37 0.99 AC 1 36 0.86 CA 1 36 0.86 CC 2 36 0.96 CCC 1 33 0.77 IGHD 3' G 11 40 0.88 GT 2 36 0.57 TC 1 36 0.86 GTT 3 33 0.0022 IGHJ T 11 42 0.042 GT 3 34 0.26 AGC 1 32 0.45 ACT 2 32 0.026 1 α equal to 0.003 A single case of significance was observed among the putative P nucleotide sequences. This was for a sequence of 3 nucleotides (GTT) which was associated with the 3' end of the IGHD region. The occurrence of 3 'GTT' sequences in the dataset remains the only significant putative P nucleotides even if the alpha value is increased to 0.01. Using a 0.05 significance level, 6 sets out of 23 appear significant, however this conclusion carries a 69% chance of being incorrect and that the results occurred by chance. Although putative P nucleotide sequences are present in 10% of sequences most of these are likely to have arisen as the result of N nucleotide addition. P nucleotides can only be confidently attributed to less than 1% of sequences with three sequences from the IgM dataset contained statistically significant P nucleotides out of the 245 IGH V-D-J rearrangements examined. The overall contribution of P nucleotides to junctional nucleotides was 9 nucleotides out of 2899 junctional nucleotides, or 0.3% of junctional nucleotides, within the IgM dataset. The probability of 'GTT' occurring within an N region is 0.007875, therefore, the sequence could be expected to occur twice at the observed position in the 245 junctions examined, by chance alone. Of the three identified P nucleotides it is therefore possible that only one is a true P nucleotide. The contribution of P nucleotides to junctional nucleotides could therefore be as low as 0.1%, with P nucleotide inclusions occurring in less than 0.5% of sequences. Discussion Investigation of the role played by nucleotide loss and addition in the generation of immunoglobulin diversity has been limited by the ability of researchers to accurately determine gene ends. The development of a statistically based partitioning method has allowed this study to gain insights into the nature of nucleotide loss and addition in the expressed human immunoglobulin repertoire. Analysis of 294 human IgM sequences revealed significant differences between average nucleotide losses from different heavy chain genes segments. IGHV genes suffer less removal in comparison to other genes, suggesting that a process or processes may act to prevent the removal of critical components or to select against sequences in which such removals have occurred. Critical components may include the conserved TGT that defines the start of the CDR3 [27] and the internal heptamer site utilized in VH gene replacement [23-25]. The extension of the analysis to the gene subgroup level showed significant differences among removals from the IGHV gene subgroups, among the IGHD subgroups at the 3' end of the D gene and between the six IGHJ gene subgroups. The most striking contrast was observed for the IGHJ gene subgroups, specifically for IGHJ3 and IGHJ6. Removals from IGHJ3 averaged only a single nucleotide, while IGHJ6 on average lost in excess of 6 nucleotides. The differences observed as part of this study suggest that the loss of nucleotides during the creation of human heavy chain immunoglobulin sequences is not random. Unique 'patterns' of exonuclease removal between gene subgroups have previously been reported in murine immunoglobulins [12,28], however we are not aware of any such reports from studies of the human repertoire. Murine IGHJ4 genes have been reported to undergo an average removal of 2 nucleotides more than any other murine IGHJ subgroup [28]. Comparison of the murine IGHJ4 sequence to that of human IGHJ6 shows these two sequences to be identical for the first 7 nucleotides. The common sequence, ATTACTA, is unique to these IGHJ subgroups. This suggests that the common sequence may be linked to the high levels of nucleotide loss experienced by these gene subgroups, relative to the other IGHJ genes. The nucleotide composition of gene has previously been stated to influence exonuclease processing in the murine system [12]. The presence of two 'motifs' was thought to be the determining factor in the outcome of exonuclease processing. One motif involves stretches of two or more G or C nucleotides and is referred to here as the S motif. The other motif is composed of stretches of three or more consecutive A or T nucleotides and is referred to here as the W motif. S motifs in murine sequences were associated with low average removals from gene region ends, while the presence of W motifs correlated with high average removals [12]. Similar results were seen for human immunoglobulins in this study, with average removals from IGHJ genes containing 5' S motifs being significantly lower than from those containing 5' W motifs. Interestingly, the average position of the first S motif within the IGHJ genes coincided with the average level of removal from IGHJ genes. The correlation between S motif position and average exonuclease removal suggests that the S motif may act to block continued exonuclease removal from the gene region end. This may explain the high removals from the human IGHJ6 and murine IGHJ4 gene segments, as these sequences lack S motifs which may prevent such extensive exonuclease processing. Consideration of the W and S motif composition of the 3' D gene segments showed the IGHD2 and IGHD3 gene subgroups to be rich in W motifs and to lack S motifs (data not shown). These two subgroups showed higher average removals compared to other IGHD subgroups. The relationship between nucleotide composition and exonuclease activity could therefore explain the significant differences observed at the 3' end of the IGHD gene subgroups. IGHD2 and IGHD3 are both long D genes. A relationship between D gene length and exonuclease activity may have therefore been acting to influence exonuclease processing. Examination of exonuclease activity of D genes grouped by length did reveal significant differences (data not shown), however, these differences were only evident at the 3' end of the D genes and as the analysis was confined to IGHD2 and IGHD3 sequences, it is difficult to conclude whether sequence length has a role. The influence of nucleotide composition on exonuclease removals from heavy chain gene is easily examined in the IGHJ genes, due to the small number of alleles and the clear division of sequences based on the presence or absence of S and W motifs. Significant differences in the V genes were not further examined as the larger number of alleles made sample groups too small to allow for meaningful statistical analysis. The absence of significant differences at the 5' end of the IGHD genes may result from the lack of distinct differences in the nucleotide composition of these sequences. This would make any variations in exonuclease processing more subtle, and thus a larger sample size would be necessary to observe any differences. P nucleotide addition has been reported to contribute to diversity in between 10% [7,8,29] and 41% [9] of immunoglobulin sequences. Initial analysis of putative P nucleotides in the dataset of 294 human IgM sequences in this study revealed the frequency the presence of putative P nucleotides to be around ten percent of sequences. This is consistent with previous reports [7,10,29]. Statistical analysis of the putative P nucleotides sequences, however, revealed that only one 'P nucleotide sequence' was observed at a frequency that was significantly above the frequency that would be expected from N nucleotide addition alone. This suggests that the true contribution of P nucleotides to diversity in the expressed human IgM repertoire is much lower than previously reported, with P nucleotides present in less than 1% of sequences and accounting for approximately 0.3% of junctional nucleotides in the IgM dataset. It should be noted that even those P nucleotides accepted on the basis of statistical analysis carry a degree of uncertainty. Accounting for the possible misidentification of TdT additions among P nucleotides suggests that the contribution of P nucleotides to junctional nucleotides may be even lower than 0.3%. The identification of P nucleotides during partitioning of immunoglobulin sequences introduces a greater margin of error than would result from their exclusion from partitioning processes. For example, the rare nature of P nucleotide inclusion in rearranged immunoglobulins means that in a dataset of 1000 sequences less than 10 sequences may possess P nucleotides. Arbitrary identification of putative P nucleotides in the same dataset could however lead to 100 of the sequences being identified as having P addition. The error is therefore smaller if P nucleotides are not allocated as part of the partitioning process. The statistical demonstration of the phenomenon of P nucleotides by Meier and Lewis utilized altered recombination substrates, where the IGHV, IGHD and IGHJ gene regions were replaced by restriction sites on a plasmid vector [8]. The recombinant substrates were then transfected into murine cell lines and the processing of the substrates was then examined. Meier and Lewis observed that putative P nucleotides occur at a significant frequency among the processed recombinant substrates and this has been used as the basis for the allocation of P nucleotides in subsequent immunoglobulin studies [8]. Examination of the frequency of P nucleotides among the recombination substrates, however, revealed a five fold greater frequency of P nucleotide inclusions than was seen among adult murine T cell receptors and immunoglobulin sequences [8] and adult human immunoglobulins [7,29]. The applicability of Meier and Lewis' statistical analysis of the altered recombination substrates to demonstrate the contribution of P nucleotides must therefore be questioned, as the reporting of statistical significance is likely to be a direct result of the elevated frequency of putative P nucleotides among the recombinant substrates. Examination of putative P nucleotides at five-fold lower frequencies eliminates the significance observed in the original studies and supports the figure reported here of a contribution to junctional diversity in less than 1% of sequences (data not shown). Conclusions Substantial in vitro evidence in support of the formation of hairpin loops as part of the immunoglobulin rearrangement mechanism [6,30,31] and for the creation of P nucleotides as part of the process of hairpin loop opening exists [32,33]. The results reported here suggest that the P nucleotides generated by the loop opening do not, however, contribute significantly to the diversity of the final rearranged immunoglobulin. Exonuclease processing of IGH genes is not random and the nucleotide composition of the gene end appears to be influential. Further investigations into factor(s) influencing the exonuclease processing of gene ends will be required in order to elucidate the exact nature of the relationship between the gene end and exonuclease processing. Methods Dataset creation Human IgM sequences were obtained from public nucleotide databases; IMGT/LIGM-DB available through the IMGT, The IMmunoGeneTics Information SystemR, [18], the Entrez nucleotide database from the National Center for Biotechnology Information (NCBI) [34] and the Kabat Database of Sequences of Immunological Interest [35]. The sequences obtained were screened to exclude those sequences of fetal origin, those associated with diseases and those of a non-productive nature. Screening was necessary to avoid the introduction of any biases that may be associated with particular disease states or stages of immunological development. Sequences that contained in excess of 5 mutations within the V gene were also excluded from the final dataset. Sequences displaying higher levels of mutation were excluded from the analysis as the partitioning method utilised is most accurately applied to sequences with low levels of mutation. Partitioning of rearranged immunoglobulin sequences The determination of genes and N regions of the sequences within the dataset was performed with the aid of a statistical analysis of point mutations [21]. This method uses the number of mutations in the core region of the V genes to predict the level of mutation within other regions of the immunoglobulin sequence. The approach is based upon the mutability of trinucleotides, while also factoring in the exponential decay of somatic point mutations [36] and the effects of antigen selection [21]. The key to the analysis is the calculation of mutability scores for the various genes. Mutability scores can be used to indicate the likelihood that mutations will be distributed in a particular way between two or more parts of an immunoglobulin sequence. The focus of the study upon exonuclease removal required careful definition of rules for the identification of nucleotide losses. Preliminary alignment of sequences were performed using IMGT/V-QUEST [18]. Where identical IMGT/V-QUEST alignment scores were achieved to different alleles of a germline gene, the allele first allele reported was recorded. IMGT/V-QUEST compares input sequences to the IMGT reference sets which are the most complete sets of sequences that are available for IGHV, IGHD and IGHJ functional genes, their alleles and open reading frame genes. Alignments produced by IMGT/V-QUEST give an overall indication of similarity between a rearranged sequence and germline genes. D gene determination was performed using previously described criteria [21], where the level of required similarity to a germline sequence was dictated by the length of the junction and the likelihood of N nucleotides being misidentified as IGHD segments by chance. To aid in the allocation of D genes, a D Gene Alignment Utility was developed. This web based tool allowed alignments to be performed between a junctional sequence and all germline D genes, including inverted D gene sequences [37] obtained from the IMGT Reference Directory [19]. The program utilized an altered Smith-Waterman algorithm that did not allow for gaps [38]. Difficulties with immunoglobulin partitioning are often experienced, especially in the determination of gene ends. In this study, runs of consecutive nucleotide differences between a IGH V-(D)-J rearrangement and a germline sequence at a gene end were always attributed to exonuclease removal, rather than mutation of the gene end. The portion of a IGH V-(D)-J rearrangement that contained such differences, with respect to the germline sequence, was allocated to the N region, and the gene was considered to have undergone exonuclease processing at the gene end. Situations where IGHV or IGHJ ends revealed a series of differences and similarities to the germline sequence required the consideration of each possible combination of exonuclease removal and point mutation that could have led to the creation of the observed region end. Probabilities were calculated for each 'path' to the observed segment end. Mutability scores for the region end were used to determine the likelihood of point mutations contributing to the region end. TdT addition probabilities were used to calculate the likelihood of N additions generating particular nucleotide sequences. The TdT probabilities used were p(G) = 0.35, p(C) = 0.35, p(A) = 0.15, p(T) = 0.15 [5]. The path that displayed the greatest likelihood was used to allocate nucleotides to either an N region or a region end. Sequences that lacked exonuclease removals from gene region end(s) were examined for the presence of P nucleotides [2]. Self-complementary repeats of the gene end located in the neighboring N region were designated as putative P nucleotides. For example, if the V gene ended with the nucleotides GA, then CT was sought at the start of the N1 region. All possible lengths of P insertions were considered as part of the identification process. Examination of P nucleotides The contribution of P nucleotides to immunoglobulin diversity was investigated through data generated by the investigations reported here. Unique putative P nucleotides were identified and their appearance was tallied within the dataset. The number of junctions that displayed a lack of exonuclease activity at one or more gene ends was also calculated. The cumulative binomial probability of observing a given number of P nucleotides or greater was then calculated for each putative P nucleotide sequence in an attempt to estimate the contribution of P nucleotides to immunoglobulin diversity. Role of W and S Motifs in exonuclease processing The effect of W and S motifs upon exonuclease activity was investigated within the IGHJ genes. The IGHJ genes were grouped based upon the relative location of such motifs. W motifs were defined as sequences of 3 or more consecutive A or T nucleotides. S motifs were defined as sequences of 2 or more consecutive G or C nucleotides. The presence of these motifs was considered within the first 15 5' nucleotides of the IGHJ genes. Three sets were established, based upon the observed configurations of the motifs in the IGHJ subgroups; S motif followed by W motif, S motif only and W motif only (Table 2). Analysis of exonuclease removals for each of these groups was then performed by calculating the average number of nucleotides removed from the IGHJ end for each set. Table 2 Grouping of IGHJ genes by relative location of W and S motifs J Gene Sequence1 Group IGHJ1*01 GCTGAATACTTCCAG 5' S only IGHJ2*01 TGCTACTGGTACTTG 5' S only IGHJ3*01 TGATGCTTTTGATGT 5' S then W IGHJ3*02 ATGCTTTTGATATCT 5' S then W IGHJ4*01 ACTACTTTGACTACT 5' W only IGHJ4*02 ACTACTTTGACTACT 5' W only IGHJ4*03 GCTACTTTGACTACT 5' S then W IGHJ5*01 ACAACTGGTTCGACT 5' S only IGHJ5*02 ACAACTGGTTCGACC 5' S only IGHJ6*01 ATTACTACTACTACT 5' W only IGHJ6*02 ATTACTACTACTACT 5' W only IGHJ6*03 ATTACTACTACTACT 5' W only 1W motifs shown underlined, S motifs shown in italics Statistical analysis The extent of nucleotide deletion was calculated as the average number of nucleotides removed for a given dataset. Significant differences between average removals were determined using one-way ANOVA for normally distributed datasets, and Kruskal-Wallis Test for other datasets. Where significant differences were found, multiple comparison testing was carried out using Tukey's Multiple Comparison Test, for normally distributed datasets, and Dunn's Multiple Comparison Test for non-normally distributed datasets. All analysis of exonuclease removals was carried out using GraphPad Prism (Version 3.00, 1999, GraphPad Software) with an alpha value of 0.05. An analysis of P nucleotides was performed by calculating the probability that putative P nucleotides may actually have resulted from N nucleotide addition by TdT. Probabilities were calculated as described by Meier and Lewis [8]. The probability of the presence of the observed or a greater number of P nucleotides was calculated as follows [8]: where, n is the observed number of P nucleotides and N is the total number of sequences containing junctional inserts equal to or greater than the length of the P nucleotide(s) being examined, and p is the expected frequency of the observed P nucleotide sequence, which was calculated using reported TdT frequencies [5]. Probabilities were calculated for each observed putative P nucleotide sequence, and the alpha value was adjusted for the number of comparisons made, using the Bonferroni correction. Authors contributions KJ carried out the dataset creation and partitioning, performed the statistical analysis and drafted the paper. BG aided in the design of the study and the development of the statistical analysis. WS advised on study design and development. AC devised the study, aided in study design and co-ordination and revised the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 294 Partitioned human IgM Sequences 294 human IgM sequences were partitioned using a statistically based model and used in the examination of exonuclease activity and P nucleotide addition in the expressed human repertoire. The file is in Microsoft Excel format. Click here for file ==== Refs Tonegawa S Somatic generation of antibody diversity. Nature 1983 302 575 81 6300689 Lafaille J DeCloux A Bonneville M Takagaki Y Tonegawa S Junctional sequence of T cell receptor gamma delta genes: implications for gamma delta T cell linages and for a novel intermediate of V-(D)-J joining. Cell 1989 59 859 870 2590942 10.1016/0092-8674(89)90609-0 Alt FW Baltimore D Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc Natl Acad Sci USA 1982 79 4118 22 6287467 Kim S Davis M Sinn E Patten P Hood L Antibody diversity: somatic hypermutation of rearranged VH genes. Cell 1981 27 573 81 6101208 10.1016/0092-8674(81)90399-8 Basu M Hegde MV Modak MJ Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase. Biochem Biophys Res Commun 1983 111 1105 12 6301484 Roth DB Menetski JP Nakajima PB Bosma MJ Gellert M V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 1992 70 983 91 1356077 10.1016/0092-8674(92)90248-B Komori T Sugiyama H N Sequences, P Nucleotides and Short Sequence Homologies at Junctional Sites in VH to VHDJH and VHDJH to JH Joining. Mol Immunol 1993 30 1393 1398 8232324 10.1016/0161-5890(93)90100-P Meier J Lewis S P Nucleotides in V(D)J Recombination: A Fine-Structure Analysis. Mol and Cell Biol 1993 13 1078 1092 8380891 Rosner K Winter DB Tarone RE Skovgaard GL Bohr VA Gearhart PJ Third complementarity-determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non-mutated genes. Immunol 2001 103 179 87 10.1046/j.1365-2567.2001.01220.x Gauss GH Lieber MR Mechanistic constraints on diversity in human V(D)J recombination. Mol Cell Biol 1996 16 258 69 8524303 Nadel B Tehranchi S Feeney A Coding end processing is similar throughout ontogeny. J Immunol 1995 154 6430 6436 7759879 Nadel B Feeney A Influence of coding-end sequence on coding-end processing in V(D) J recombination. J Immunol 1995 155 4322 4329 7594591 Nadel B Feeney A Nucleotide deletion and P addition in V(D)J recombination: a determinant role of the coding-end sequence. Mol Cell Biol 1997 17 3768 3778 9199310 Feeney AJ Comparison of junctional diversity in the neonatal and adult immunoglobulin repertoires. Int Rev Immunol 1992 8 113 22 1602207 Yamada M Wasserman R Reichard B Shane S Caton A Rovera G Preferential Utilization of Specific Immunoglobulin Heavy Chain Diversity and Joining Segments in Adult Human Peripheral Blood B Lymphocytes. J Exp Med 1991 173 395 407 1899102 10.1084/jem.173.2.395 Giudicelli V Lefranc MP Ontology for immunogenetics: the IMGT-ONTOLOGY. Bioinformatics 1999 15 1047 54 10745995 10.1093/bioinformatics/15.12.1047 Lefranc MP Giudicelli V Ginestoux C Bosc N Folch G Guiraudou D Jabado-Michaloud J Magris S Scaviner D Thouvenin V Combres K Girod D Jeanjean S Protat C Yousfi-Monod M Duprat E Kaas Q Pommie C Chaume D Lefranc G IMGT-ONTOLOGY for immunogenetics and immunoinformatics. In Silico Biol 2004 4 17 29 15089751 Lefranc MP IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 2003 31 307 10 12520009 10.1093/nar/gkg085 LeFranc MP LeFranc G The Immunoglobulins FactBook. Academic Press, London 2001 458 Lefranc MP Pommie C Ruiz M Giudicelli V Foulquier E Truong L Thouvenin-Contet V Lefranc G IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003 27 55 77 12477501 10.1016/S0145-305X(02)00039-3 Collins AM Ikutani M Puiu D Buck GA Nadkarni A Gaeta B Partitioning of rearranged Ig genes by mutation analysis demonstrates D-D fusion and V gene replacement in the expressed human repertoire. J Immunol 2004 172 340 8 14688342 Souto-Carneiro MM Longo NS Russ DE Sun HW Lipsky PE Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER. J Immunol 2004 172 6790 802 15153497 Kleinfield RW Weigert MG Analysis of VH gene replacement events in a B cell lymphoma. J Immunol 1989 142 4475 82 2498430 Gay D Saunders T Camper S Weigert M Receptor editing: an approach by autoreactive B cells to escape tolerance. J Exp Med 1993 177 999 1008 8459227 10.1084/jem.177.4.999 Radic MZ Zouali M Receptor editing, immune diversification, and self-tolerance. Immunity 1996 5 505 11 8986711 10.1016/S1074-7613(00)80266-6 Zhang Z Wang YH Zemlin M Findley HW Bridges SL Burrows PD Cooper MD Molecular mechanism of serial VH gene replacement. Ann NY Acad Sci 2003 987 270 3 12727651 Lefranc MP Nomenclature of the human immunoglobulin heavy (IGH) genes. Exp Clin Immunogenet 2001 18 100 16 11340299 10.1159/000049189 Feeney AJ Victor KD Vu K Nadel B Chukwuocha RU Influence of the V(D)J recombination mechanism on the formation of the primary T and B cell repertoires. Semin Immunol 1994 6 155 63 7948955 10.1006/smim.1994.1021 Lieber MR Chang CP Gallo M Gauss G Gerstein R Islas A The mechanism of V(D) J recombination: site-specificity, reaction fidelity and immunologic diversity. Semin Immunol 1994 6 143 53 7948954 10.1006/smim.1994.1020 Lieber MR Site-specific recombination in the immune system. FASEB J 1991 5 2934 44 1752360 McBlane JF van Gent DC Ramsden DA Romeo C Cuomo CA Gellert M Oettinger MA Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 1995 83 387 95 8521468 10.1016/0092-8674(95)90116-7 Lewis SM The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol 1994 56 27 150 8073949 Lewis S P Nucleotide Insertions and the Resolution of Hairpin DNA Structures in Mammalian Cells. PNAS 1994 91 1332 1336 8108412 Benson DA Karsch-Mizrachi I Lipman DJ Ostell J Wheeler DL GenBank: update. Nucleic Acids Res 2004 32 D23 6 14681350 10.1093/nar/gkh045 Kabat EA Wu TT Perry HM Gottesman SK Foeller C Sequences of Proteins of Immunological Interest. US Dept of Health and Human Services, Bethsuda, MD 1991 Rada C Milstein C The intrinsic hypermutability of antibody heavy and light chain genes decays exponentially. EMBO J 2001 20 4570 6 11500383 10.1093/emboj/20.16.4570 Jackson KJL The D Gene Alignment Utility. 2003 Smith TF Waterman MS Identification of common molecular subsequences. J Mol Biol 1981 147 195 7 7265238

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==== Front BMC NeurosciBMC Neuroscience1471-2202BioMed Central London 1471-2202-5-281532095310.1186/1471-2202-5-28Research ArticleFingersomatotopy in area 3b: an fMRI-study van Westen Danielle [email protected] Peter [email protected] Johan [email protected]én Birgitta [email protected] Göran [email protected] Elna-Marie [email protected] Dept of Diagnostic Radiology, Lund University Hospital, 221 85 Lund, Sweden2 Cognitive Neurophysiology, MR Research Centre, N8, Dept of Clinical Neuroscience, Karolinska University Hospital, 171 76 Stockholm, Sweden3 Dept of Hand Surgery, University Hospital, 205 02 Malmö, Sweden2004 20 8 2004 5 28 28 26 1 2004 20 8 2004 Copyright © 2004 van Westen et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The primary sensory cortex (S1) in the postcentral gyrus is comprised of four areas that each contain a body map, where the representation of the hand is located with the thumb most laterally, anteriorly and inferiorly and the little finger most medially, posteriorly and superiorly. Previous studies on somatotopy using functional MRI have either used low field strength, have included a small number of subjects or failed to attribute activations to any area within S1. In the present study we included twenty subjects, who were investigated at 3 Tesla (T). We focused specifically on Brodmann area 3b, which neurons have discrete receptive fields with a potentially more clearcut somatotopic organisation. The spatial distribution for all fingers' peak activation was determined and group as well as individual analysis was performed. Results Activation maps from 18 subjects were of adequate quality; in 17 subjects activations were present for all fingers and these data were further analysed. In the group analysis the thumb was located most laterally, anteriorly and inferiorly with the other fingers sequentially positioned more medially, posteriorly and superiorly. At the individual level this somatotopic relationship was present for the thumb and little finger, with a higher variability for the fingers in between. The Euclidian distance between the first and fifth finger was 17.2 mm, between the first and second finger 10.6 mm and between the remaining fingers on average 6.3 mm. Conclusion Results from the group analysis, that is both the location of the fingers and the Euclidian distances, are well comparable to results from previous studies using a wide range of modalities. On the subject level the spatial localisation of the fingers showed a less stringent somatotopic order so that the location of a finger in a single subject cannot be predicted from the group result. ==== Body Background The first somatotopic maps of the homuncular organisation of the primary somatosensory cortex (S1) were established in 1937 by using intra-operative electrical stimulation of the brain surface [1]. Subsequent, non-invasive investigations in humans on the hand representation in S1 have described a somatotopic organisation along the central sulcus with the thumb located laterally, anteriorly and inferiorly to the little finger [2-6]. Studies in non-human primates have revealed the cytoarchitectonic subdivisions of S1, namely areas 3a, 3b, 1 and 2, that outline the cortex in the postcentral gyrus [7]. Area 3a occupies the fundus of the central sulcus, area 3b the anterior wall of the postcentral gyrus, area1 its crown and area 2 its posterior wall. Each area contains a fairly complete map of the body surface and is the cortical representation of different somatosensory receptors. In area 3b the neurons are predominantly responsive to stimulation of cutaneous receptors. As opposed to neurons in area 1, that also receive input from cutaneous receptors, those in area 3b possess discrete receptive fields with a homuncular organisation that may be more distinct [8]. Previous studies on somatotopy in the hand area using functional Magnetic Resonance Imaging (fMRI) have yielded varying results. Gelnar et al. failed to show a somatotopy in S1 when applying vibratory stimuli to three of the fingers of the right hand [5]. Maldjian et al. demonstrated somatotopy in 3 out of 5 subjects [3]. Similarly, Kurth et al., using electrical stimulation of two fingers, found somatotopically arranged activation patterns in 5 out of 20 subjects [9]. In a follow-up study where activation of all fingers in area 3b was found in 7 out of 10 subjects, the same authors reported a general somatotopy, without further specification [4]. Methods differ considerably between these three studies with regard to anatomical considerations, number of subjects studied, and the field strength used. Maldjian et al. used the highest field strength, 4 Tesla (T), while both Kurth et al. and Gelnar et al. used 1.5 T [3-5]. However, Maldjian et al. did not contribute activations to any area in S1. Also Maldjian et al. included the smallest number of subjects (5); data from one were discarded due to motion artefacts and group analysis was based on data from the remaining 4. In the present study we readdressed the issue of somatotopy in the hand area as assessed with fMRI. Our aim was to optimise results by including a larger number of subjects, by focussing on area 3 b, where homuncular organisation expectedly is most distinct and by performing fMRI at 3 Tesla (T). Results Tactile stimulation of the fingers of the dominant hand yielded activation in contra-and ipsilateral S1, contra- or bilateral secondary sensory cortex (S2), ipsilateral cerebellum, and in some subjects the contralateral thalamus. Significant activation for all five fingers in area 3b was present in seventeen subjects and these data were further analyzed. In the one volunteer excluded from the analysis, activation was present for four fingers. The spatial distribution of the activations in the contralateral S1 for tactile stimulation versus rest in one subject is shown in Figure 2. A somatotopic organisation with the representation of the thumb located laterally to the little finger was present in 16 out of 17 subjects, with the thumb located anteriorly to the little finger in 14 out of 17 subjects and with the thumb located inferiorly to that of the little finger in 16 out of 17 subjects. Group averages of the distances from D2 to D1 (D2-D1), D3 to D1(D3-D1), D4 to D1 (D4-D1) and D5 to D1 (D5-D1) are presented in the Table and shown as graphs in Figure 3. Combined these indicate a strict somatotopy with the distance to D1 increasing for every finger in each of the three directions. Distances to D1 were compared for neighbouring fingers. In the medial-lateral direction, the distance D4-D1 was different from D3-D1. In the anterior-posterior direction a significant difference was observed between D4-D1 and D3-D1. Finally, in the superior-inferior direction the distances D2-D1 and D3-D1 as well as D3-D1 and D4-D1 differed; the location of D2, as determined by its distance to D1, was different from the location of D1 [0, 0, 0]; no difference was found between the distances D5-D1 and D4-D1. Considering that the three coordinates [x, y and z] together define one point in the 3D Cartesian space, the coordinates of D3, D4 and D5, differed from those of D1, p now <0.05/3, corrected for multiple comparisons (not in Table). The Euclidian distance from D1 to D2 was 10.6 mm (SEM ± 1.5). The distance from D2 to D3 was 5.5 mm (± 0.9), from D3 to D4 7.4 mm (± 1.1) and from D4 to D5 6.8 mm (± 1.2), resulting in an average for D2-D3, D3-D4 and D4-D5 of 6.6 mm. The spatial extension of the representation of the hand in area 3b, defined as the Euclidian distance between D1 and D5 was 17.2 mm (± 2.0 mm). Discussion In the group average from the present study the strict somatotopic organisation in the primary sensory cortex known from studies using a variety of modalities, was reproduced [2-6]. The fingers' average activations were laid out on the body map, with the thumb located most laterally, anteriorly and inferiorly and the little finger most medially, posteriorly and superiorly and the remaining fingers in between, the distance to the thumb increasing for every finger in each of the three directions. In individual subjects the arrangement in the hand representation with the thumb located laterally, anteriorly and inferiorly to the little finger is frequently found, while the remaining fingers may or may not display the orderly lateral-to-medial, anterior-to-posterior and inferior-to-superior organisation 'D1-D2-D3-D4-D5' [2,3,10,11]. We chose to present group averages as it is our belief our results would have greater significance if a regular somatotopy was present at the group level. Body maps in non-human primates demonstrating the regular sequence mentioned above, were established with cortical single unit recordings and are supposedly the golden standard. The present study is based on data from 20 subjects, generating results available for analysis from 18 of these; the spatial representation of all fingers in area 3b of the primary somatosensory cortex was localised in 17 subjects. The average extension of the hand representation in area 3b of 17 mm with a somatotopic arrangement of fingers 1-5 as described above is consistent with results from previous studies using a range of modalities [2-4,7]. Also the mean distance between D2-D3, D3-D4 and D4-D5 of 6.6 mm and 6.3 mm for main and differential effects, respectively, is in good agreement with human electrophysiological and fMRI data [2,4]. The larger distance between the thumb and index finger as compared to distances between subsequent fingers suggests a larger representation for the thumb. This finding is in agreement with results from a study using electrocorticography with subdural electrodes in three patients [1,12]. With the spatial resolution used in this study, 3 × 3 × 3 mm3, resampled to 1.5 × 1.5 × 1.5 mm3, activation for all five fingers was found in 17 of 18 subjects. For comparison, Kurth et al. used a resolution of 1.7 × 1.7 × 1.7 mm3 and electrical stimulation with ring electrodes and found activation in area 3b for all five fingers in 7 out of 10 subjects (70%) [4]. The present study showed activations for all fingers in 94 % of subjects. This difference might be due to the higher field strength used in this study as the higher magnetisation vector and sensitivity to changes in susceptibility increase the signal-to-noise ratio. In one subject excluded from the analysis, activation was present for four fingers and another was excluded due to general lack of activation. Lack of activation may be due to subjects being 'low-activators' in fMRI experiments, due to inadequate stimulation or to some other, unknown factor. The type of stimulation used is decisive of what area in S1 can be expected to be activated. For example, neurons in area 3a are responsive to deep receptor and proprioceptive stimulation and in one study punctate tactile stimulation did not activate area 3a [13]. Also, receptive fields are maximally focused in area 3b, while in area 1 receptive fields become larger and more complex. In area 2, receptive fields are even more complex with reduplications. This combined knowledge made area 3b the area of our choice to study somatotopy and explains why we chose not to report on activations in areas 3a, 1 and 2. We found activation in the anterior wall of the postcentral gyrus, defined as area 3b according to our operational definition during tactile stimulation (Fig 1). More pronounced activation was noticed frequently in the crown or posterior wall of the postcentral gyrus, defined as areas 1 and 2 (Fig 2). Similar observations have been made in other studies using both fMRI and PET [5,14]. According to studies in non-human primates the representation of the distal fingertips in area 1 points posteriorly, a finding confirmed in a recent fMRI-study on humans [15]. Area 2 then is the mirror-image of area 1 with the fingertips pointing anteriorly. The activation in the posterior wall might represent activation in both areas 1 and 2 localised at their meeting point, i.e. the fingertips. The larger cluster size of this activation is explained by the clusters arising from area 1 and 2 being contiguous and therefore additive. In the present study the activation of area 1 and 2 is probably due to hierarchical processing in the rostrocaudal direction within S1. A previous observation that electrical stimulation of the cutaneous afferents of the median nerve resulted in evoked potentials in area 3b after 30 msec while a potential in area 1 was seen after another 5 msec lends support to this assumption [16]. Conclusion In the group analysis, a somatotopic organisation for all the fingers in the hand representation of area 3b could be demonstrated using fMRI; the Euclidian distance between the thumb and the little finger was well comparable to that determined in previous studies. On the subject level the cortical somatosensory representation of the thumb was located laterally, anteriorly, and inferiorly to that of the little finger in 14 out of 17 subjects. The spatial localisation of the remaining fingers showed a less stringent somatotopic order when compared individually. Methods Subjects Twenty healthy, self-reportedlly right-handed volunteers (6 male and 14 female, age 21–43 years, mean 29.4 years) were included in the study. The protocol was approved by the local ethics committee and written informed consent was obtained. All volunteers had normal images on a fluid-attenuated inversion recovery (FLAIR) sequence. Stimulation Tactile stimulation consisted of brushing the glabrous skin of the two distal phalanges of each finger continuously forwards and backwards with a commercially available tooth brush. During the experiment, volunteers were positioned on the MR table with their right arm from the elbow down in a padded cast, that also provided support for the dorsal part of the hand. They were instructed to rest their arm against the magnet bore so that both arm and hand were relaxed. Pieces of soft cloth were placed between the fingers in order to avoid that stimulation also involved a neighbouring finger. The frequency was 1 Hz; no forced pressure was exerted. Consistency was tested on a finger model firmly taped onto a computerized electronic scale (Biopac Systems, DA 100B, MP 100A; Macintosh Powerbook G3 with software AcqKnowledge 3.5): the intra-examiner error was 18% based on a mean pressure of 6.64 g, standard deviation 1.199 g. Imaging MRI was performed using a 3 T head scanner (Siemens Allegra) with a quadrature birdcage coil. Morphological T1-weighted images with a resolution of 1 × 1 × 1 mm3 using a magnetisation prepared gradient echo sequence (MPRAGE) were acquired. Functional echo-planar image volumes of the whole brain (number of slices = 49, thickness = 3 mm, voxel size = 3 × 3 × 3 mm3) sensitised to the Blood Oxygenation Level Dependent (BOLD)-effect (echo time = 30 ms) were acquired. Five scanning sessions were performed. Each session included 92 functional volumes with a temporal resolution of 3 seconds. The first two volumes in each session were discarded from further analysis to allow for initial T1-equilibrium effects. Experimental protocol Fingers 1 to 5 (D1 = thumb, D5 = little finger) of the right hand were stimulated sequentially in separate sessions according to a block design that included four periods of stimulation and five of rest for each finger. The epoch length for both stimulation and rest periods was 30 seconds. Postprocessing Image processing and analysis were carried out using the SPM99 soft ware package [17]. All functional images were resliced to a voxel size of 1.5 × 1.5 × 1.5 mm3 and then realigned to the first image and coregistered to the T1-weighted image volume. All data were spatially filtered using an isotropic 4 mm, full-width, half-maximum Gaussian kernel. A high pass filter (cut off frequency 0.008 Hz) was applied to eliminate low frequency signal fluctuations. In order to preserve each subject's somatotopic arrangement in area 3b no normalization to a common brain atlas was performed. Data analysis Functional data from two subjects were excluded due to major motion artefacts (subject no. 7) and global lack of activation (subject no.18). Task specific effects were estimated using the general linear model (GLM) that included a box car function convolved with the canonical hemodynamic response function in SPM99. The effect of sensory stimulation of each finger versus rest was determined using a one-sample t-test of pertinent linear contrasts of parameter estimates in each subject with a significance level of p < 0.001 (uncorrected). Then the spatial coordinates of the peak activation voxel in area 3b were determined. Due to lack of neuroanatomical landmarks, exact delineation of the cytoarchitectonically defined areas within S1 cannot be achieved in MR images. Therefore we used an operational definition based on cytoarchitectonic studies of S1 on 10 post-mortem brains. In >50 % of these brains area 3a was located in the fundus of the central sulcus, area 3b in the rostral bank of the postcentral gyrus and area 1 on its crown reaching down into the postcentral sulcus [18,19]. Fig 1 illustrates these locations, that continue along the central sulcus. As interareal borders vary across brains, the same authors constructed probability maps for each area by superimposing histological volumes of the individual brains on a computerized reference brain. Volumes of interest (VOIs) were defined for each area in which >50 % of the brains had a representation of that area. Despite close relationship of areas 3a, 3b and 1 in the postcentral gyrus, the three VOIs overlapped by <1% of their volumes. These probability maps were at hand when the spatial coordinates of the peak activation voxel in area 3b were determined. Activated peak voxels were labelled as belonging to area 3b when they were located within the anterior wall of the postcentral gyrus (Figure 1). The spatial coordinates of the peak voxel for the thumb (D1) was defined as being at origo [0, 0, 0] in a 3D Cartesian coordinate system.When the spatial coordinates for all fingers were known in all subjects, somatotopy was assessed by determining the average distances for the whole group from each finger to the thumb. Euclidian distances between fingers as well as from each finger to the thumb (D1) were calculated as: with xDI, yDI and zDI representing the coordinates of the finger DI and xDII, yDII and zDII representing the coordinates of DII in the three directions x, y, z in a system where the coordinates of D1 are at origo [0, 0, 0]. Statistics Each finger's distance to the first finger was compared to that of the directly neighbouring fingers using the Wilcoxon matched pairs test with a significance level of p < 0.05. The Euclidian distances to the first finger were compared for each finger using the Wilcoxon matched pairs test with a significance level of p < 0.05. Authors' contributions DvW conceived of the study, performed the image analysis and drafted the manuscript. PF guided the image analysis. JO designed the MR-parameters. EML participated in the design of the study. BR and GL initiated and conducted the stimulation consistency study. All authors read and approved the final manuscript. Acknowledgments The authors thank dr S Geyer, Research Center Jülich, Germany for probability maps delineating areas 3a, 3b and 1. This study was supported by the Swedish Medical Research Council, the Swedish Brain Foundation, the Medical Faculty at Lund University, the Skåne County Council Research and Development Foundation and the Royal Physiographic Society in Lund.. Part of this work was presented at the annual meeting of the European Society for Magnetic Resonance in Medicine and Biology, Rotterdam 2003. Figures and Tables Figure 1 Areas of S1 as defined in cytoarchitectonic studies on 10 post-mortem brains [18, 19]: area 3a occupies the fundus of the central sulcus (dark blue), area 3b the anterior wall of the postcentral gyrus (red), area1 its crown (light blue) and area 2 its posterior wall (green). The black arrow indicates the central sulcus. Figure 2 Activation in the contralateral somatosensory cortex during tactile stimulation of the fingers of the right hand versus rest in a single subject. The first column shows transverse anatomical image with z-coordinate indicated. Subsequent columns show the activation patterns in S1 overlayed on magnified T1-weighted images for each finger. The location of the peak voxel in area 3b is indicated by blue crosshairs. Figure 3 Distance to the thumb (D1) for each finger (mm), mean (diamonds) and SEM (errorbars), as presented in Table. The coordinates for D1 are defined as origo [0, 0, 0]. Distances to D1 are shown in the medial-lateral (M-L) direction, in the left panel also in the posterior-anterior (P-A) direction and in the right panel also in the superior-inferior (S-I) direction. The fingers are sequentially positioned more medially, posteriorly and superiorly. Table Distance to finger 1 Finger x L → M y A → P z I → S Euclidian D1 0 (0) 0 (0) 0 (0) 0 (0) D2 2.9 (1.6) 0.4 (1.8) 4.8 (1.0) 10.6 (1.5) D3 3.6 (1.4) 1.4 (1.5) 6.8 (1.2) 11.2 (1.5) D4 6.9 (1.4) 4.6 (1.7) 9.4 (1.4) 15.4 (1.7) D5 7.4 (1.7) 6.8 (1.2) 10.4 (2) 17.2 (2.0) Distance from each finger (D2, D3, D4, D5) to the thumb (D1) ± standard error of the mean (SEM) (mm). The lateral-to-medial direction is named 'x', the anterior-to-posterior direction 'y' and the inferior-to-superior direction 'z'. Underlined are those distances to D1 that differ significantly from the distance of the previous finger to D1 (Wilcoxon matched pairs test, p < 0.05). ==== Refs Penfield W Boldrey E Somatic motor and sensory representation in the cerebral cortex of man as studied by electrical stimulation Brain 1937 60 389 443 Hari R Karhu J Hamalainen M Knuutila J Salonen O Sams M Vilkman V Functional organization of the human first and second somatosensory cortices: a neuromagnetic study Eur J Neurosc 1993 5 724 734 Maldjian JA Gottschalk A Patel RS Detre JA Alsop DC The sensory somatotopic map of the human hand demonstrated at 4 Tesla NeuroImage 1999 10 55 62 10385581 10.1006/nimg.1999.0448 Kurth R Villringer K Curio G Wolf KJ Krause T Repenthin J Schwiemann J Deuchert M Villringer A fMRI shows multiple somatotopic digit representations in human primary somatosensory cortex NeuroReport 2000 11 1487 1491 10841363 Gelnar PA Krauss BR Szeverenyi NM Apkarian AV Fingertip representation in the human somatosensory cortex: an fMRI study NeuroImage 1998 7 261 283 9626668 10.1006/nimg.1998.0341 Buchner H Fuchs M Wischmann HA Dossel O Ludwig I Knepper A Berg P Source analysis of median nerve and finger stimulated somatosensory evoked potentials: multichannel simultaneous recording of electric and magnetic fields combined with 3D-MR tomography Brain Topogr 1994 6 299 310 7946929 Kaas JH Nelson RJ Sur M Lin CS Merzenich MM Multiple representations of the body within the primary somatosensory cortex of primates Science 1979 204 521 523 107591 Kaas JH Multiple representations of the body within the primary somatosensory cortex of primates Physiol Rev 1983 63 206 231 6401864 Kurth R Villringer K Mackert BM Schwiemann J Braun J Curio G Villringer A Wolf KJ fMRI assessment of somatotopy in human Brodmann area 3b by electrical finger stimulation NeuroReport 1998 9 207 212 9507956 Mogilner A Grossman JA Ribary U Joliot M Volkmann J Rapaport D Beasley RW Llinás RR Somatosensory cortical plasticity in adult humans revealed by magnetoencephalography Proc Natl Acad Sci 1993 90 3593 3597 8386377 Baumgartner C Doppelbauer A Sutherling WW Zeitlhofer J Lindinger G Lind C Deecke L Human somatosensory cortical finger representation as studied by combined neuromagnetic and neuroelectric measurements Neurosci Lett 1991 16 103 8 10.1016/0304-3940(91)90518-X Sutherling WW Levesque MF Baumgartner C Cortical sensory representation of the human hand: size of finger regions and nonoverlapping digit somatotopy Neurology 1992 42 1020 1028 1579225 Moore CI Stern CE Corkin S Fischl B Gray AC Rosen BR Dale AM Segregation of somatosensory activation in the human rolandic cortex using fMRI J Neurophysiol 2000 84 558 69 10899227 Burton H MacLeod AM Videen TO Raichle ME Multiple foci in parietal and frontal cortex activated by rubbing embossed grating patterns across fingerpads: a positron emission tomography study in humans Cereb Cortex 1997 7 3 17 9023428 10.1093/cercor/7.1.3 Blankenburg F Ruben J Meyer R Schwiemann J Villringer A Evidence for a rostral-to-caudal somatotopic organization in human primary somatosensory cortex with mirror-reversal in areas 3b and 1 Cereb Cortex 2003 13 987 993 12902398 10.1093/cercor/13.9.987 Allison T McCarthy G Wood CC Darcey TM Spencer DD Williamson PD Human cortical potentials evoked by stimulation of the median nerve. I. Cytoarchitectonic areas generating short-latency activity J Neurophysiol 1989 62 694 710 2769354 Statistical Parametric Mapping Geyer S Schleicher A Zilles K Areas 3a, 3b and 1 of Human primary Somatosensory cortex, 1. Microstructural organisation and interindividual variability NeuroImage 1999 10 63 83 10385582 10.1006/nimg.1999.0440 Geyer S Schormann T Mohlberg H Zilles K Areas 3a, 3b and 1 of Human primary Somatosensory cortex, 2. Spatial normalisation to standard anatomical space NeuroImage 2000 11 684 696 10860796 10.1006/nimg.2000.0548

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-301534502910.1186/1471-2334-4-30Technical AdvanceA robust, low- to medium-throughput prnp genotyping system in sheep Buitkamp Johannes [email protected] Jördis [email protected] Bavarian State Research Center for Agriculture, Institute of Animal Breeding, Prof.-Dürrwaechter-Platz 1, 85586 Poing, Germany2004 2 9 2004 4 30 30 25 5 2004 2 9 2004 Copyright © 2004 Buitkamp and Semmer; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background In many countries breeding programs for resistance to scrapie in sheep are established. Therefore, the demand on genotyping capacities of the polymorphisms of the prion protein gene (prnp) relevant to presently known disease associations and EU regulations is steadily increasing. Most published typing methods are not well suited for routine typing of large sample numbers in smaller service laboratories for different reasons: they require partly manual data processing, sophisticated and sensitive protocols, high efforts regarding time and manpower, multiple step reactions or substantial hardware investments. To overcome these drawbacks, we developed a prnp typing method that is based on a `multiplex amplification refractory mutation system' (ARMS) reaction. Methods In this study we combined the amplification refractory mutation system (ARMS) with standard fluorescent based fragment length analyses method to develop a prnp genotyping method (PRNP ARMS). Results By optimised primer design it was possible to type the 4 relevant single nucleotide polymorphisms (SNPs) in the prnp simultaneously in one multiplex reaction. Automated fragment length analysis enabled automated allele designation. Suitability of the PRNP ARMS for routine application was proven by typing samples with known genotypes and larger sample numbers from half-sib families. Conclusion The ARMS PRNP typing method established in this study is universally suited for a broad range of typing projects with different requirements. It provides an efficient and inexpensive diagnostic mutation analysis that will improve the quality of prnp genotyping compared with other low-cost methods. It can be implemented by most molecular genetic laboratories using standard equipment. ==== Body Background Scrapie is a contagious prion disease [1] of sheep and goat. In contrast to BSE, there is no evidence for a transmission to humans. Nevertheless, BSE is experimentally transmittable to sheep and the resulting disease can not be distinguished from scrapie [2,3]. Even though BSE has not been found in farmed sheep yet, the possibility that BSE occurs in "scrapie" diseased sheep can not be excluded. Therefore, scrapie control programs were implemented in many countries. Since no vaccine or therapeutic means is currently available, these programs rely on selective breeding for scrapie resistance. Susceptibility to scrapie is largely controlled by three polymorphic amino acid positions (136, 154, 171) of the ovine prion protein gene (prnp) [4] and reliable genotyping of the corresponding DNA polymorphism is required as a basis for selection decisions. For determination of prnp alleles or haplotypes, there are several typing techniques applied today. Direct sequencing covering the region of exon 2 encoding amino acid positions 136, 154 and 171 is the most accurate method, that enables the typing of additional and detection of hitherto unknown polymorphisms. Other methods are classical PCR-restriction fragment length polymorphism (RFLP)-typing [5,6], DNA strand conformation polymorphism detection, denaturing gradient gel electrophoresis (DGGE) [7], PCR-single-strand conformational polymorphism (PCR-SSCP) [8], hybridisation with allele-specific oligonucleotides [9,10], primer extension [11], and so on. For high-throughput genotyping matrix assisted laser desporption/ionisation – time of flight (MALDI-TOF) systems (e.g. for bovine prnp [12] and the LGC-web page [13]), taqman® (Applied Biosystems, CA) [14], and pyrosequencing (Biotage AB, Uppsala, Sweden) are used, but methodologically details of these methods are not very well documented in the literature. The amplification refractory mutation system (ARMS) method is well established [15] and has been applied not only to single nucleotide polymorphism (SNP) detection but also to haplotype determination [16]. In the present work we combined the use of fluorescence labelled oligonucleotides with ARMS technology for the simultaneous detection of 4 SNPs without using common primers. Based on this PRNP ARMS method we developed a cost-effective, well-reliable and low- to high-throughput technology for prnp typing that can easily be adopted by a wide range of other laboratories. Methods DNA-Samples EDTA-blood or Typifix®-tissue samples were collected from half-sib families from three common sheep breeds in Bavaria. All lambs were born in the years 2001 and 2002. DNA was isolated by using the E.Z.N.A. Blood DNA Mini Kit (#12-3482-03, PEQLAB Biotechnologie GmbH, Erlangen, Germany) or the NucleoSpin® Multi-96 Tissue kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), respectively. PRNP-ARMS Design of Primers An overview of primer locations is given in Fig. 1. Initially, allele-specific primers were designed that differed only at the 3'-nucleotide (given in bold letters in Table 1). Using these primers it was not possible to obtain reliable discrimination of alleles. Especially the simultaneous use of primers for alleles Q-171 and R-171 resulted in cross-amplification products. Therefore, a number of additional primers were tested to optimise allele discrimination. Destabilizing mismatches were introduced at the first or second penultimate base in addition to the allele-specific base at the 3' termini of the primers (ARMS principle, indicated by small letters in Table 1). The sense primers specific for the alleles at amino acid position 136 were labelled with two different fluorochromes, 4, 7, 2', 7'-tetrabetweenchloro-6-carboxyfluorescein (TET) and 6-carboxyfluorescein (FAM) (Table 1, Fig. 1). That allows the detection and discrimination of the alleles by standard gel electrophoresis on a fluorescent sequencer. Pieces of neutral sequence [17] were added to the 5'-end (underlined in Table 1) of the unlabeled antisense primers for amino acid positions 154 and 171. These enable the differentiation of the alleles by different lengths. Mismatches between primers were deliberately introduced at the 5' region in these neutral sequences to avoid jumping amplification products. For a delineation of primer sequences with a reference sequence (Fig. 3). PCR-reaction For the ARMS reaction haplotypes were amplified from 2 μl of DNA solution with standard buffer conditions, 1.5 mM MgCl2, dNTP's (25 nM each), and 0.75 units of HotStar-taq polymerase (Qiagen, Hilden, Germany) in a final volume of 10 μl on a t-gradient 96-well thermocycler (Biometra, Göttingen, Germany). Primer concentrations were 10 nmol each. Cycling was for 15 min at 95°C, [0.5 min at 94°C, 1 min at 62°C, 1 min at 74°C]33× without final extension. Fragment analysis Fragment lengths of ARMS amplification products were analysed on an ABI PRISM® 310 genetic analyser (Applied Biosystems, Foster City, CA) with 5 sec injection time, 15 kV, 60° and 18 min runtime using 36 cm capillaries. Allele designations were generated automatically using the Genotyper® software (V. 2.5, Applied Biosystems) as described [18]. The data were imported into an Microsoft Access database. Standard DNA samples representing different alleles were included in the assay to control each PCR mix and reaction. As an additional control a sql-query was used to flag all genotypes transferred to the database that were not compatible with the 15 known standard genotypes (e.g. peaks from FAM in the range of 153 bp) for retyping. Comparative sequencing Part of prnp exon 3 coding for amino acids 47 to 246 of the prion protein was amplified in 10 μl reaction volume using 2 μl of genomic DNA, 0.05 μM of each primer (5'-tcc tgg agg caa ccg cta tc-3' and 5'-gga gga tca cag gag ggg aag-3'), 50 μM of each dNTP, 0.5 units of HotStar-taq polymerase (Qiagen, Hilden, Germany), and reaction buffer containing 1.5 mM MgCl2. Cycling-conditions on a Biometra T gradient 96-well thermocycler were: 15 min at 95°C, [0.5 min at 95°C, 1 min at 60°C, 0,5 min at 72°C]35× and 10 min, 60°C final extension. Before direct sequencing PCR-reactions were purified using the 96-well MultiScreen-PCR plates (Millipore, Bedford, MA). Sequencing was performed using BigDye V 2.0 terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing analysis was run on an ABI PRISM® 310 genetic analyser. Results Development of the PRNP ARMS method We have established a multiplex `amplification refractory mutation system' (ARMS) based method for the identification of prnp 136-154-171 genotypes in sheep. The PRNP ARMS method was developed using standard DNAs representing all five main haplotypes. In a first stage, different primer pairs were tested for allele specific amplification using an anneal-temperature gradient. By redesign of primers for amino-acid positions 154 and 171 and introducing deliberate mismatches close to the 3' end an optimised primer set was established (Table 1) allowing multiplex, allele-specific amplification. "False positive fragments" (peaks above the background signal generated by primers complementary to alleles that were not present in the corresponding sample) were not observed using the optimised primer set. Nevertheless, the average peaks heights differed between alleles. For example the average peak heights of the allele Q-154 was 1.5 times higher than that of R-154. The ratio of peak heights in heterozygous individuals differed from 0.5 to 2.5 fold (compare Fig. 2). These were observed when different PCR assays with low number of samples and small volumes of master-mix are compared. The peak height differences depend mainly on the relative primer concentrations and can be avoided e.g. by using premixed primer solutions containing all ARMS primers. For automated "base calling" the minimum peak height was set to 100 (in practice most samples gave peak heights well above 1000). Proof-of-principle To provide a proof-of-principle the same 140 sheep DNAs were analysed by direct PCR sequencing and by PRNP ARMS. Typing results were identical with both methods. In a second step, 420 sheep from half-sib families and 20 samples from the international sheep and goat DNA typing comparison test 2003 of the International Society of Animal Genetics were analysed by the PRNP ARMS method. All genotypes followed the rules of Mendelian inheritance and no deviation from the main 5 haplotype patterns was observed. Allele frequencies derived from these animals from three different Bavarian breeds are shown in Table 2. Paternity of all lambs was checked with a set of 9 microsatellites (data not shown). Typical electropherograms representing different haplotype combinations are shown in Fig. 2. Allele length determination was highly reproducible (Table 3). The range of individual peaks from each individual allele was well below +/-1 bp allowing unambiguous allele-calling and automatic generation of results tables using the genotyper software. Furthermore, the PRNP ARMS reaction worked fairly stable with different DNA qualities. The quality and concentration of the DNAs that were isolated from tissue varied widely (5 – 50 ng/μl, various degrees of fragmentation). Failed PCR reactions (e.g. one primer missing) were identified by analysing the standard samples. Failed individual samples (e.g. due to low DNA concentrations or failure of the fragment analyses run) were easily identified by missing or very low and out-of-range peaks. Nevertheless, the proportion of samples that had to be retyped was well below 1%. Discussion The PRNP ARMS genotyping method has several advantages. It is based on a one step reaction using competitive allele discrimination. The fragments can be analysed on an automated sequencer that allows data to be directly transferred to a database. The reaction proved to be highly specific and no false negative or positive results were observed yet. Furthermore, it is robust with respect to variations of DNA quality and, since no further purification or reaction is necessary, the costs for consumables are low. The ARMS method allows the determination of partial (136-154 and 136-171, compare Fig. 1) prnp haplotypes, thereby facilitating the detection of 'complex' prnp genotypes that do not match one of the 15 standard genotype patterns. If necessary, it can easily be extended to complete haplotype determination by adding additional labelled forward primers for position 154 (for haplotype 154-171). All methods currently available for SNP typing are inherent sensitive to nucleotide changes within the primer attachment or restriction sites. The resulting mismatches can cause inconclusive typing results or null alleles, as frequently observed with microsatellite loci. Therefore, it is crucial to be aware of this phenomenon and to take measures to reduce the risk of mistyping. The primers for PRNP ARMS were carefully checked against a prnp in-house database containing 16 published and 11 unpublished (mainly from rare German and Spanish breeds) alleles. No known polymorphism interfere with the attachment of the selected ARMS primers. Nevertheless, the recently described rare polymorphism K-171 [19] with unknown effect to scrapie resistance is not included in the standard PRNP ARMS set (that gives Q-171 as typing result for this allele) and requires one additional specific oligonucleotide. This oligonucleotide should be added if the respective breeds (mainly hair breeds [19]) are genotyped. When further polymorphisms associated with resistance to scrapie will be identified appropriate oligonucleotides can easily be added. Unlabelled additional oligonucleotides can be used when SNPs upstream of the amino acid position 136 shall be typed. Nevertheless, the inclusion of positions downstream of position 136 would require additional labelled oligonucleotides. A third fluorescent dye might be useful especially when the lengths of the fragments interfere with on of the other alleles. The PRNP ARMS method is highly flexible with respect to scale and instrumentation. It is easily adoptable from low- to medium-throughput typing system using identical reaction conditions. In principle, all primer combinations can even be performed as single reactions that can be analysed on agarose gels (data not shown). Nevertheless, the reliability of genotyping results improves when competitive reaction conditions and commonly available DNA sequencing machines are used. Since all reactions can be performed in microtiter plates, the ARMS reaction can easily be adopted for a pipetting robot. Together with the one step reaction and the automated data transfer, the risk of cross-contamination or interchanging of samples is minimized. The runtime of 18 min results in 25 min analyses time per sample on the ABI 310 and could be optimised by using shorter capillaries. When using the standard run conditions on the smallest, single capillary sequencer the throughput is limited to about 55 samples per working day but can be scaled up to more than 5000 samples per day by using a 96-well capillary sequencer. That should be sufficient even for large, existing breeding programs. PRNP ARMS can be multiplexed with other typing systems (microsatellite or SNP), since it is analysed using standard fragment analysis technique. Moreover, it is suitable for further applications as e.g. allele frequency estimation from pooled DNA [20] for investigating rare breeds. Conclusions An easy and robust one-step prnp typing method has been established that is universally suited for a broad range of typing projects with different requirements. This typing method was developed by optimising ARMS primers and combining these with standard fragment length analyses. The method provides an efficient and inexpensive diagnostic mutation analysis that can be implemented by most molecular genetic laboratories using standard equipment. It should contribute to reliable and economic genotyping of the ovine prnp by the many smaller labs throughout Europe. Competing interests The authors declare that they have no competing interests. Authors' contributions J.B. designed the project and protocols involved, performed analysis of results and drafted this manuscript. J.S. conceived the ARMS reaction, fragment analysis and DNA sequencing. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We would like to thank Georg Mendel, Albert Steiner and Max Wagenpfeil for collecting sheep samples. Figures and Tables Figure 1 Scheme of PRNP ARMS-PCR design. PrP amino acid positions are given above. The allele-specific up-primers (discrimination of A-V at codon 136) are fluorescence labelled, whereas down primers (discrimination of R-H and Q-R-H at codon 154 and 171, respectively) are of different lengths (tails are shown as red lines). Figure 2 Electropherograms from 5 PRNP genotypes. The "green" lanes correspond to TET labelled fragments, the "blue" lines to FAM labelled products. The PRNP genotype of each individual is given above each lane. Only the informative lanes (no peaks were observed in the "blue" lane of A136 and no peaks were observed in the "green" lane of V136 homozygotes, respectively) are shown. The scheme at the bottom shows how the peaks relate to the alleles. Figure 3 Location of ARMS Primers within the reference sequence. Primer sequences are given above the sequence representing the "wild-type" allele (Genbank accession number AJ000739). The 5 prime and 3 prime ends are indicated. Small letters indicate deliberately introduced mismatches; underlining indicate pieces of neutral sequence that were deliberately added to elongate the primer. Table 1 ARMS primers used to type the ovine PRNP Name Sequence of Primer 5'-3' Allele Length/Label Fragment Length PRNP_136A ATACATGCTGGGAAGTGC A136 18 bp/TET - PRNP_136V CTACATGCTGGGAAGTGT V136 18 bp/FAM - PRNP_154R ACTAACGGTACATGTTTTCAC R154 21 bp/- 92 bp PRNP_154H AGTTTGTAACGGTACATGTTTTgAT H154 25 bp/- 96 bp PRNP_171Q GGAAGTTGTTCTGGTTACTATcCT Q171 24 bp/- 146 bp PRNP_171R TCAGTTAAGTTGTTCTGGTTACTATtCC R171 28 bp/- 150 bp PRNP_171H TTCTGAGCATAAAGTTGTTCTGGTTACTATAA H171 32 bp/- 154 bp Small letters indicate deliberately introduced mismatches (ARMS principle); underlining indicate pieces of neutral sequence that were added to elongate the primer to allow allele-discrimination by capillary electrophoresis. Table 2 PRNP allele frequencies (%) as observed in three sheep breeds common in Bavaria breed Allele ML SK SU AHQ 7.68 0.00 1.96 ARH 0.18 0.00 0.00 ARQ 82.14 24.58 27.45 ARR 9.82 69.49 66.67 VRQ 0.18 5.93 3.92 ML, Merinolandschaf; SK, German Blackheaded Mutton; SU, Suffolk. Table 3 Statistics for peak lengths of the PRNP ARMS system Calculated peak sizes# Dye Oligonucleotide Fragment length Mean StdDev Min Max TET PRNP_154R 92 bp 89.16 0.12 88.91 89.70 PRNP_154H 96 bp 92.13 0.09 91.98 92.45 PRNP_171Q 146 bp 142.91 0.12 142.67 143.48 PRNP_171R 150 bp 148.05 0.18 146.72 148.42 PRNP_171H 154 bp 153.13 0.72 152.31 153.74 FAM PRNP_154R 92 bp 89.56 0.13 89.36 89.84 PRNP_154H 96 bp np np np np PRNP_171Q 146 bp 143.29 0.11 143.13 143.48 PRNP_171R 150 bp np np np np PRNP_171H 154 bp np np np np #peak lengths as calculated using the genotyper software from all genotype results in the database; np: not present in the dataset ==== Refs Prusiner SB Prions Proc Natl Acad Sci USA 1998 95 13363 13383 9811807 10.1073/pnas.95.23.13363 Baylis M Houston F Kao RR McLean AR Hunter N Gravenor MB BSE – a wolf in sheep's clothing? Trends Microbiol 2002 10 563 570 12564992 10.1016/S0966-842X(02)02477-0 Foster JD Parnham D Chong A Goldmann W Hunter N Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats Vet Rec 2001 148 165 171 11258721 Hunter N Piper LR, Ruvinsky A Molecular Biology and Genetics of Scrapie in Sheep The Genetics of Sheep 1997 CAB International 225 240 Hunter N Foster JD Benson G Hope J Restriction fragment length polymorphisms of the scrapie-associated fibril protein (PrP) gene and their association with susceptibility to natural scrapie in British sheep J Gen Virol 1991 72 1287 1292 1675248 Hunter N Goldmann W Benson G Foster JD Hope J Swaledale sheep affected by natural scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie J Gen Virol 1993 74 1025 1031 8099602 Belt PB Muileman IH Schreuder BE Bos-de Ruijter J Gielkens AL Smits MA Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie J Gen Virol 1995 76 509 517 7897344 Marcos S Calvo JH González C Serrano M Haplotype determination and new variants within the ovine prion cds region Proceedings of the International Workshop on Major Genes and QTL in Sheep and Goat: 8–11 dec. 2003; Toulouse; CD-ROM communication n°2-30 2003 Hunter N Moore L Hosie BD Dingwall WS Greig A Association between natural scrapie and PrP genotype in a flock of Suffolk sheep in Scotland Vet Rec 1997 140 59 63 9023905 Ishiguro N Shinagawa M Onoe S Yamanouchi K Saito T Rapid analysis of allelic variants of the sheep PrP gene by oligonucleotide probes Microbiol Immunol 1998 42 579 582 9776400 Zsolnai A Anton I Kühn C Fesus L Detection of single-nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis Electrophoresis 2003 24 634 638 12601731 10.1002/elps.200390074 Humeny A Schiebel K Seeber S Becker CM Identification of polymorphisms within the bovine prion protein gene (Prnp) by DNA sequencing and genotyping by MALDI-TOF-MS Neurogenetics 2002 4 59 60 12030333 10.1007/s10048-001-0126-0 LGC awarded genotyping contract as part of National Scrapie Plan Cawthraw S Nadales EP Martin TC PrP genotyping in sheep using the 5' nuclease (Taqman) PCR assay Res Vet Sci 2001 70 24 Newton CR Graham A Heptinstall LE Powell SJ Summers C Kalsheker N Smith JC Markham AF Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) Nucleic Acids Res 1989 17 2503 2516 2785681 Lo YM Patel P Newton CR Markham AF Fleming KA Wainscoat JS Direct haplotype determination by double ARMS: specificity, sensitivity and genetic applications Nucleic Acids Res 1991 19 3561 3567 1852606 Lindblad-Toh K Winchester E Daly MJ Wang DG Hirschhorn JN Laviolette JP Ardlie K Reich DE Robinson E Sklar P Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse Nature Genet 2000 24 381 386 10742102 10.1038/74215 Hall JM LeDuc CA Watson AR Roter AH An approach to high-throughput genotyping Genome Res 1996 6 781 790 8889547 Guo X Kupfer DM Fitch GQ Roe BA DeSilva U Identification of a novel lysine-171 allele in the ovine prion protein (PRNP) gene Anim Genet 2003 34 303 305 12873221 10.1046/j.1365-2052.2003.01019.x Germer S Holland MJ Higuchi R High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR Genome Res 2000 10 258 266 10673283 10.1101/gr.10.2.258

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-311534743010.1186/1471-2334-4-31Research ArticleAn economic analysis of premarriage prevention of hepatitis B transmission in Iran Adibi Peyman [email protected] Mohammadreza [email protected] Delnaz [email protected] Negar [email protected] Shahin [email protected] Mohammad Hossein [email protected] Saeed [email protected] Mohammad Reza [email protected] Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, Tehran, Iran2004 4 9 2004 4 31 31 27 4 2004 4 9 2004 Copyright © 2004 Adibi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background To assess the economic aspects of HBV (hepatitis B virus) transmission prevention for premarriage individuals in a country with cultural backgrounds like Iran and intermediate endemicity of HBV infection. Methods A cost-effectiveness analysis model was used from the health care system and society perspectives. The effectiveness was defined as the number of chronic HBV infections averted owing to one of the following strategies: 1) HBsAg screening to find those would-be couples one of whom is HBsAg positive and putting seronegative subjects on a protection protocol comprising HBV vaccination, single dose HBIG and condom protection. 2) HBsAg screening as above, in addition to performing HBcAb screening in the HBsAg negative spouses of the HBsAg positive persons and giving the protocol only to HBcAb negative ones. Sensitivity and threshold analyses were conducted. Results The cost of each chronic infection averted was 202$ and 197$ for the strategies 1 and 2, respectively. Sensitivity analysis showed that strategy 2 was always slightly cheaper than strategy 1. The discounted threshold value for the lifetime costs of chronic liver disease, above which the model was cost saving was 2818$ in strategy 1 and 2747$ in strategy 2. Conclusions Though premarriage prevention of HBV transmission in the countries with cultural backgrounds similar to Iran seems cost saving, further studies determining precise costs of HBV infection in Iran can lead to a better analysis. ==== Body Background Hepatitis B is an important health problem and a major cause of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Approximately 30% of the world population (1.8 billion people) have the serologic evidence of HBV infection of whom 350 million are estimated to suffer from chronic HBV (hepatitis B virus) infection; at least 500,000 chronically infected people die of liver malignancy and cirrhosis each year [1]. According to Iranian studies, about 22% to 37% of general population in Iran are HBcAb positive [2,3] (e.g. previous exposure to HBV) and about 1.3% to 8.69% of the population are chronic HBV carriers [2-6]. Compared to the United States where HBV is the cause of 25% of chronic hepatitis cases, HBV accounts for up to 70% to 80% of chronic hepatitis cases in Iran [7]. Therefore, HBV alone is the leading cause of chronic liver disease (CLD) in Iran and it is evident that HBV transmission prevention can be one of the health priorities in the country. We believe that major routes of HBV transmission, local epidemiological factors and the already performed prevention programs of each region are among important factors in identifying the populations at risk of the infection and planning the region-specific prevention strategies. In Iran, universal neonatal vaccination against HBV started in 1993 according to WHO recommendations. It means that Iranians 10 years of age or older at the time of this study received no prevention services against HBV and so most of them could contract the infection if attacked by the virus. For this at-risk population, several preventive strategies can be suggested which of course should have economic justification. Premarriage transmission prevention can be considered as one of the possible solutions to protect this population though it should not be regarded as the only or the best solution available. Sexual contact is one of the common routes of HBV transmission. In Iran, due to a particular cultural and religious background, homosexuality is not known as a common phenomenon compared to the western countries. For the same reasons, it is very unlikely for an individual to have sexual contact (especially in the form of intercourse) with his/her would-be spouse. On the other hand, almost all premarriage individuals (those considering legal marriage) are obliged by Iranian law to undergo a predefined battery of screening tests in government-designated laboratories; this can make premarriage individuals an accessible group for a preventive intervention. Finally, since most premarriage individuals are in young age groups, they have a rather long life expectancy, which allows enough time for them to suffer from chronic complications of HBV infection in productive years of life. We decided to perform this study to provide health policy makers in Iran and those countries with similar demographic conditions (especially in the Middle East) with an economic analysis of premarriage prevention of hepatitis B transmission. Methods Model The economics of performing two rather similar strategies in addition to no intervention strategy were compared using a decision tree (Fig. 1). The software used for analysis was Decision Analysis by TreeAge (DATA™, Williamstown, MA, USA). As shown in Fig. 1, the overall options available for premarriage individuals can be one of the major strategies mentioned below: 1. Screening all premarriage individuals for HBsAg and then performing the following prevention protocol (marked as P.P. in Fig. 1) for HBsAg negative individuals whose would-be spouse is HBsAg positive: a. Three-dose HB vaccine (0, 1, 6 mo) b. Single dose HBIG injection c. Using condoms (2 boxes/mo) during all intercourses for 7 months d. Measurement of HBsAb (hepatitis B surface antibody) 1 month after the 3rd dose of the vaccine e. An extra dose vaccine and additional condom protection for another month for the persons whose HBsAb is not in protective ranges (lower than 10 IU/l) No protection protocol is considered for those couples who are both HBsAg positive or negative. 2. Screening all premarriage individuals for HBsAg followed by rescreening of the HBsAg negative spouses of HBsAg positive persons for HBcAb and finally performing the prevention protocol (e.g. the a to e steps above) only for HBcAb (and HBsAg) negative individuals whose would-be spouse is HBsAg positive. No protection protocol is considered for those couples who are both HBsAg positive or negative. 3. No screening and no prevention. The main analysis considered the perspective of health care system. However, in the final analysis (including the sensitivity analysis), a threshold analysis was performed from societal perspective. The results were expressed by the cost per chronic infection (e.g. more than 6 months HBsAg positive) averted (e.g. average cost effectiveness). Assumptions The assumptions in the model included: 1) HBV vaccination is not harmful for the individual or community; 2) the efficacy of preventive methods such as 3-dose HB vaccine, HBIG injection and condoms in preventing sexual transmission of HBV do not significantly vary by geographic region; 3) the compliance of the population for receiving the preventive methods is 100%; 4) the preventive methods are available throughout the country and can cover 100% of the population; 5) HBcAb positive persons are not at risk of HBV infection and this group will not be considered for receiving preventive measures; 6) all HBsAg positive individuals are HBcAb positive too; 7) the sensitivity and specificity of the screening tests (for HBsAg and HBcAb) are 100%; 8) the average age at marriage is 25 years of age for both sexes; 9) the costs associated with CLD are incurred during a 10-year period starting at the age of 50 [1,24]; 10) the transmission rate of HBV from men to women and vice versa are equal; 11) premarriage individuals do not have intercourse before marriage; and 12) Iran is located in a region with intermediate prevalence of hepatitis B. Probabilities and costs The probabilities included in the decision analysis model (Table 1) were assumed to be of 2 types: 1) the probabilities that do not significantly vary by geographic location (e.g. the efficacy of 3-dose HB vaccine, HBIG injection and condom in preventing sexual transmission of HBV) and 2) the ones that seem to be significantly different in various geographic locations (e.g. HBsAg or HBcAb prevalence rates in the society). To have preliminary estimates of the latter category probabilities, all available and accurate Iranian medical literature (published from 1993 to 2003) were reviewed. For the former category probabilities, international resources (PubMed) were included in addition to the Iranian sources mentioned above. If a probability was not found in medical literature, the consensus of an expert team including 5 gastroenterologists collaborating with our research center was considered as the base case value. A review of Iranian studies showed that the prevalence of HBsAg in general population varied from 1.2% to 8.69% in different parts of Iran [2-6]. In this study, we considered an average rate of 2% as the baseline in a range of 1% to 9% (Table 1). The Iranian studies also showed that the prevalence of HBcAb varied from 15% to 37% in different parts of Iran. Therefore, we assumed an average rate of 20% as the baseline in a range of 15% to 40% (Table 1) [2,3]. The review of international and Iranian medical literature revealed that the probability of being HBsAg positive for an HBsAg positive person's spouse (P3) is about 4% to 15% [8-11]. We assumed a baseline probability of 5%. (Table 1) The probability of becoming HBsAg positive for an HBsAg positive person's spouse after receiving the prevention protocol (P4) was indirectly calculated by the formula below: P4 = P3 (1 - efficacy of preventive protocol) The baseline value for the efficacy of the prevention protocol used in our model to protect against spouse-spouse HBV transmission (3-dose HB vaccine, HBIG injection and condom protection up to complete immunity) was assumed to be 90%. The figure was reached by considering the values found in the literature for the efficacy of 3-dose HB vaccine [12,13], HBIG injection [14-19] and condom protection [20-23] which was finally modified by consensus from the expert team described above. The most pessimistic and optimistic estimations for the efficacy of the prevention protocol were assumed to be 75% and 100%, respectively. Therefore, the baseline value for P4 was assumed to be 0.05% in a range of 0% to 1.25%. The direct medical costs of interventions (Table 2) were extracted from the resources and tariffs of Iranian Health Ministry, Iran Pasteur Institute and Iranian Transfusion Organization in 2003 (unpublished data) and were used as baseline costs in the model. The indirect medical costs of the intervention such as transportation and time costs for the recipients of the preventive methods (to receive the services) were assumed to be zero and were not included in the model. The costs of the averted morbidity (HBV infection especially chronic liver disease) were not directly put in the model because of unavailability of relative Iranian studies. However, as a solution to better analysis, the latter costs were calculated as a final variable in a threshold analysis, considering the fact that of the adults in chronic carrier state, 15% will eventually develop chronic liver disease (CLD) [1,24,25]. In most economic analyses, the costs are modified for the outcomes occurring in the future, a process called discounting. Considering the average age at marriage and the age at which CLD starts (see assumptions) the cost of CLD calculated through the threshold analysis was discounted by a discount rate of 3% to the beginning of the 10-year period of CLD development. The costs are expressed in the text and tables in US $ and Iranian Rials (1US $ = 8300 Iranian Rials). The currency conversion rate reported here is the one for mid-2003 when the study was performed. Sensitivity analysis Uncertainty management, which is one of the central processes in decision-making, usually involves working with probabilities that usually vary in different circumstances. Therefore, the outcome values and final decision is prone to change when the value of probabilities change. Sensitivity analysis is a method in which the final decision and the value of outcomes are estimated while each probability (univariate) or combinations of probabilities (multivariate) are varied in a reasonable range. This will reveal the variables whose change the model is sensitive to. In this study, univariate sensitivity analysis was performed for prevalence of HBsAg positivity in general population (P1), prevalence of HBcAb positivity in general population (P2), probability of becoming HBsAg positive for an HBsAg positive person's spouse (P3) and probability of becoming HBsAg positive for an HBsAg positive person's spouse after receiving the prevention protocol (P4). Multivariate sensitivity analysis was performed for P1 and P2, considering the fact that the two probabilities were dependent. All of the direct medical costs of the intervention (Table 2) were assumed to be constant in the sensitivity analyses. The results of the above sensitivity analyses were evaluated in respect of their impact on the value of the cost per chronic HBV infection averted and the preference of strategies 1 and 2. Results Having run the model for baseline values (Tables 1 and 2), the average cost effectiveness of strategies 1 (without additional screening for HBcAb) and 2 (including additional screening for HBcAb) were 1,675,500 Rials (202 $) and 1,633,200 Rials (197 $) for each chronic HBV infection prevented, respectively. The worst-case analysis (e.g. setting all input probability values so that they would act to decrease effectiveness and increase the costs) was performed setting P1, P2 and P3 at their minimum values and P4 at its maximum value. It showed that the average cost-effectiveness ratio of strategy 1 would be 2,460,204 Rials (296 $) and that of strategy 2 would be 2,440,015 Rials (293 $) in the worst case. It is noteworthy that the costs were not varied and were kept at their baseline values in this sensitivity analysis. A threshold analysis was performed to find the threshold value for the lifetime cost of CLD for one individual (from a societal perspective) below which the interventions in the model were not cost saving (e.g. the net benefit was negative). The analysis was performed keeping all other variables at their baseline values. The preliminary (non-discounted) threshold value for the lifetime cost of CLD was found to be 11,170,000 Rials (1346 $) and 10,888,000 Rials (1312 $) using strategies 1 and 2, respectively. After discounting, the threshold figures for CLD costs were 23387500 Rials (2818 $) for strategy 1 and 22797054 Rials (2747 $) for strategy 2. This showed that for the cost of CLD higher than the thresholds above, the respective strategies used for HBV transmission prevention would be cost saving. Sensitivity analysis Sensitivity analysis showed that when the prevalence of HBsAg positivity in general population (P1) varied from the minimum to maximum, the cost per chronic HBV infection averted varied from 1,503,440 Rials (183 $) to 2,740,560 Rials (330 $) in strategy 1 and from 1,482,620 Rials (179 $) to 2,568,310 Rials (309 $) in strategy 2 (Fig. 2). When the prevalence of HBcAb positivity in general population (P2) increased from the lowest to highest, the cost per chronic HBV infection prevented did not vary in strategy 1; but in strategy 2, it decreased from 1,645,720 Rials (198 $) to 1,561,530 Rials (188 $). Therefore, the higher rates of HBcAb positivity made the cost of strategy 2 become remarkably lower than that of strategy 1 (Fig. 3). Changing the probability of becoming HBsAg positive for an HBsAg positive person's spouse after marriage (P3) from minimum to maximum varied the cost-effectiveness ratio from 2094370 Rials (252 $) to 558,500 Rials (67 $) in strategy 1 and from 2,041,500 Rials (246 $) to 544,400 Rials (66 $) in strategy 2. It shows that higher spouse-to-spouse transmission rates significantly increase the cost of both strategies (Fig 4). When the probability of becoming HBsAg positive for an HBsAg positive person's spouse after receiving prevention protocol (P4) changed from the highest to lowest, the cost-effectiveness ratio decreased from 2,010,600 Rials (242 $) to 1,507,950 Rials (182 $) in strategy 1 and from 1,959,840 Rials (236 $) to 1,469,880 Rials (177 $) in strategy 2. It shows that higher efficacy of the preventive protocol results in lower costs-effectiveness ratios (Fig. 5). Strategy 2 was always cheaper than strategy 1 for all values of P1, P2, P3 and P4 in the univariate sensitivity analyses (explained above). The results of multivariate sensitivity analysis of the two variables P1(the prevalence of HBsAg positivity in population) and P2 (the prevalence of HBcAb positivity in population) revealed that the strategy 2 was always cheaper than strategy 1 while the two probabilities varied. Discussion Preventing sexual transmission of HBV is not a new issue; however, the authors did not encounter any studies directly addressing the economic aspects of premarriage prevention of hepatitis B in their literature review. The reason can be the particular cultural backgrounds of Iranian community in which extramarital sexual relationships with the would-be spouse is expected to be rare due to strong traditional and religious bans against it. Consequently, the model used in this study may not be an appropriate one for countries with remarkable cultural difference in terms of extramarital sexual relationship such as Western countries. On the other hand, the cultural similarities between Iran and some other Eastern countries can increase the external validity of our model for policy makers in such countries. One of the important challenges in our study was the lack of precise data regarding some probabilities. Most of the studies in our literature review contained the prevalence of HBsAg positivity (chronic HBsAg carrier state) in the spouses of HBsAg positive people and the extent to which it was different from the rate in general population [8-11]. Because such a rate existed in literature, we preferred to use this prevalence rate representing the initial outcome of sexual contact with an HBsAg positive spouse after marriage. Thus, what is seen in the model (Fig. 1) as HBsAg+ final outcome does not mean getting infected with HBV; it means getting into a chronic HBsAg carrier state. In our model, we assumed all such cases to be chronic HBsAg carriers that entered the chronic carrier state asymptomatically or following an acute infection; 15% of such carriers could finally develop CLD during their lifespan [1,24,25]. Therefore, it is obvious that our model have primarily focused on more chronic outcomes of HBV infection (e.g. we ignored the costs of acute infections). However, this will not endanger the data robustness in our study; instead, it will always guarantee that all of the cost-effectiveness ratios calculated here are actually higher than that would be resulted with including the costs for acute cases which usually comprise a considerable portion of the symptomatic cases in adults [1,24,25]. The costs of acute and chronic liver disease (CLD) due to HBV infection in Iran were other variables for which data was lacking. One of the important reasons we did not use a Markov model to calculate CLD costs for Iran was the lack of important necessary data for running a Markov model (e.g. lack of data on age specific mortality rates, etc.). We first calculated cost-effectiveness ratios ignoring the costs of acute and chronic liver disease (the costs of the morbidity averted); this surely exaggerated the calculated cost-effectiveness ratios in the study. Thus, one should judge them from a more optimistic point of view. In the second step, to compensate for the lack of lifetime CLD costs in Iranian literature, we performed a threshold analysis using the baseline values for input probabilities and determined a threshold value for the lifetime costs of CLD in Iran, above which the preventive interventions were cost saving. The threshold levels for CLD lifetime costs estimated above do not seem high costs compared with the costs that CLD can impose on the society in terms of direct and indirect medical costs and productivity losses due to time spent sick or years of life lost because of premature death. The relevant medical costs or the costs associated with the productivity losses due to CLD was not accessible at the time of this research, so we discussed the point through some indirect comparisons considering the threshold we calculated for lifetime CLD costs. Though comparing the costs of CLD in Iran with that in the United States does not seem a standard approach, the large differences between the threshold figure we calculated for CLD costs in Iran and the CLD costs estimations (including productivity losses) mentioned in the studies for the United States in 2001 (64,382 $) [25] may partially reveal some facts [1,24,25]. To perform a more realistic comparison, we used Purchasing Power Parity (PPP) rates instead of exchange rates to convert the threshold cost in Rials into PPP dollars. PPP is defined as the numbers of units of a country's currency needed to buy in the country the same amounts of goods and services as, say, one US dollar would buy in the United States. The PPP rate for Iranian Rial was extracted from the National Health Account 2002 by Planning and Management Organization of Iran (unpublished data) and from figures reported by the World Bank Group. The threshold cost using PPP rates would be equal to about 9615 to 9863 PPP $ (for 2 strategies). Therefore, even if the total CLD costs in a country like Iran were 7-fold smaller than that in the United States, the strategies 1 and 2 mentioned in the model could still be cost saving. To give a sketchy view of some of CLD costs in Iran, the costs of a liver biopsy and those of a pharmacotherapy regimen related to CLD was retrieved by contacting discharge and accounting departments of a state-run hospital and a major drug distributor in Tehran. The discounted cost for an uncomplicated liver biopsy needing 1 to 2 days of hospitalization, blood coagulation serial tests and a special liver biopsy needle turned out to be about 2,093,778 Rials (252 $) in all state-run hospitals in Iran. Pharmacotherapy with new antiviral and immunomodulatory drugs is employed for treatment of active chronic liver disease in patients with hepatitis B [26]. The discounted cost of lamivudine, a typical example of these drugs, can be another instance of costs CLD imposes on many patients. A 12-month course of lamivudine (Iranian brand) in Iran can cost a patient 2,521,956 Rials (304 $). If the same calculation is performed for the foreign brand of lamivudine available in Iran (Zeffix), the discounted cost will be 12,991,892 Rials (1565 $). In addition, these costs may be much higher when some more expensive drug regimens are used or more prolonged regimens are repeated due to chronicity or intractability of disease. The costs mentioned here can not directly give a clue to total CLD costs and mentioning them was to help the reader get a view of the scale of some familiar CLD costs in Iran. In a different approach, we converted the threshold value into the number of productive months for a national of a country having a GDP (Gross National Product) per capita like Iran. GDP per capita shows the amount that an individual contributes to domestic income every year. According to the World Bank Group, Iran had a GDP per capita of 1641 $ in 2002, and the average annual growth of GDP per capita in Iran for 2002 to 2006 was 4.5%. From here, the GDP per capita in 2003 comes to be 1790 $. Assuming that the average growth of GDP per capita for Iran will remain at 5% in the coming 25 years (the period used for discounting the CLD threshold costs), the GDP per capita will be 6062 $ at the end of the period. Considering such figures, the threshold calculated above would be equal to about 6 months of productivity based on a GDP view. The average number of Quality Adjusted Life Years (QALY) that CLD can deduct of an Iranian's life is not yet determined. Nevertheless, a 6-month period does not seem a long time compared with the life years lost due to premature mortality and the QALYs lost due to sickness in the proportion of CLD patients with cirrhosis and hepatocellular carcinoma only. Finally, the fact that the strategies would be cost effective can be further emphasized when taking into account the costs of acute cases of HBV infection that comprise the majority of symptomatic cases in adults and were ignored in the model due to lack of Iranian data and for sake of simplicity. Another similar topic that merits discussion here is the topic of mother-to-child vertical transmission of HBV. If a female gets into a chronic HBsAg carrier state and remains positive for HBeAg during pregnancy, it is very likely that her child is infected with HBV. Since a considerable proportion of infected infants will get chronic HBV carriers, this can lead to newer CLD cases further increasing the costs of CLD. Considering this fact, the cost of averted morbidity owing to the preventive strategies will increase even more and the model would seem more cost-effective. On the other hand, the prevention protocol in our model might seem a bit extravagantly designed when looking at the final HBsAb test and the extradose of HB vaccine administered when HBsAb serum levels are insufficient. In addition, one may argue that condoms may be more available while being nearly as protective against HBV transmission as HBIG injection. This can be useful in modifications poor countries can make to the model to get similar results in lower price or with more flexible/available choices. The average cost-effectiveness ratios associated with the two preventive strategies shown in the model did not differ much. The strategy 2 was always slightly cheaper than strategy 1. When the prevalence of HBcAb positive people in the general population (P2) rose, strategy 2 would get remarkably cheaper. Finally, it is noteworthy to mention the issue of compliance. As explained in methods, we assumed all recipients of the preventive interventions (strategies 1 and 2) would be 100% compliant and there was a full coverage of such services in the country. The reason for such assumption was the strict regulations set by Iranian government for all premarriage individuals to undergo a battery of screening tests in which the strategies in our model could be integrated. Nevertheless, we can assume that compliance variations can affect the efficacy of our prevention protocol (e.g. with lower rates of compliance for accepting preventive strategies, we will have lower efficacy of the prevention protocol). As stated in results, we performed sensitivity analysis for P4 (probability of becoming HBsAg positive for an HBsAg positive person's spouse after receiving the prevention protocol), which is a variable dependent on the efficacy of the prevention protocol (see the formula in methods). Therefore, we indirectly incorporated compliance into our model's sensitivity analysis. Considering compliance issue, we may prefer strategy 1 because it includes fewer steps (e.g. it does not include screening for HBcAb) and may be easier to administer in a low compliance population especially that it is negligibly more expensive than strategy 2. Conclusions Finally, we conclude that applying the preventive strategies in our model for HBVsexual transmission prevention before marriage in the countries with cultural backgrounds similar to Iran seems cost saving. Further investigations in the country for precise calculation of costs of HBV infection especially the costs associated with CLD is necessary for more accurate economic evaluations. Competing Interests Nond declared. Author's Contributions PA: Proposing the main idea, supervising the project and counseling the methodology development MR: Developing methodology, critical appraisal, rewriting the final article from draft, literature review, and responding to reviewers DR: Literature review, methodology development, decision tree development, calculations and results, and writing the first draft of the article NB: Contributing to decision tree development, searching literature for costs SA: Review literature for probabilities, contributing to decision tree MHS: Counseling the scenarios of the decision tree model SS: Counseling the methodology, model development and discounting problems MRZ: Senior supervisor of the research project All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The research project was accomplished in and financially supported by Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. The authors wish to express their special gratitude to Dr Ahmad Shavvakhi for his kind assistance and counseling in this study. Figures and Tables Figure 1 The decision tree model for premarriage prevention of HBV sexual transmission. Two similar intervention strategies start with HBsAg screening of all premarriage individuals. HBsAg negative individuals whose would-be spouse is HBsAg positive are considered for further intervention. Strategy 2 contains additional screening for HBcAb and applying the preventive protocol (P.P.) -see text- only to those negative for HBcAb (assumed to be serosusceptible) whereas in strategy 1, no HBcAb screening occurs and the prevention protocol (P.P.) is immediately performed. Only important probabilities are depicted in the figure. P1, P2, P3, P4 are defined in Table 1. P.P. stands for the preventive protocol explained in methods section in text. Figure 2 While the prevalence of HBsAg positivity in general population (P1) varied from the minimum to maximum in sensitivity analysis, the cost per HBV chronic infection averted varied from 1,503,440 Rials (183 $) to 2,740,560 Rials (330 $) in strategy 1 and from 1,482,620 Rials (179 $) to 2,568,310 Rials (309 $) in strategy 2; strategy 2 remained slightly cheaper throughout the range. Figure 3 When the prevalence of HBcAb positivity in general population (P2) increased from the lowest to highest limit, the cost per chronic HBV infection prevented did not vary in strategy 1, but in strategy 2, it decreased from 1,645,720 Rials (198 $) to 1,561,530 Rials (188 $). Therefore, the higher rates of HBcAb positivity made the cost of strategy 2 become more remarkably lower than that of strategy 1. Figure 4 Changing the probability of becoming HBsAg positive for an HBsAg positive person's spouse (P3) after marriage from minimum to maximum varied the cost per HBV chronic infection averted from 2094370 Rials (252 $) to 558,500 Rials (67 $) in strategy 1 and from 2,041,500 Rials (246 $) to 544,400 Rials (66 $) in strategy 2. It shows that higher spouse-to-spouse transmission rates significantly increase the cost of both strategies. Figure 5 When the probability of becoming HBsAg positive for an HBsAg positive person's spouse after receiving prevention methods (P4) changed from the highest to lowest, the cost per HBV chronic infection averted decreased from 2,010,600 Rials (242 $) to 1,507,950 Rials (182 $) in strategy 1 from 1,959,840 Rials (236 $) to 1,469,880 Rials (177 $) in strategy 2. It shows that higher efficacy of the preventive intervention results in lower costs-effectiveness ratios. Table 1 Input values for the probabilities in the model. Min. and Max. values were used in sensitivity analysis. Variable Definition Baseline Min. Max. References P1 Prevalence of HBsAg positivity in general population 0.02 0.01 0.09 2–6 P2 Prevalence of HBcAb positivity in general population 0.20 0.15 0.40 2,3 P3 Probability of becoming HBsAg + for an HBsAg + person's spouse 0.05 0.04 0.15 8–11 P4 Probability of becoming HBsAg + for an HBsAg + person's spouse after receiving prevention protocol § 0.005 0 0.0125 12–23 § See text for details. Table 2 Input values for the costs in the model.§ Variable Definition Cost Rials (US $)* C1 Cost of HBsAg screening 30000 (3.6) C2 Cost of HBcAb screening 30000 (3.6) C3 Cost of HB vaccination (one dose) 40000 (4.8) C4 Cost of HBIG injection (single dose) 120000 (14.5) C5 Cost of Condoms (one box) 8000 (1) C6 Cost of HBsAb screening 30000 (3.6) § The direct medical costs of the interventions were extracted from the resources and tariffs of Iranian Health Ministry, Iran Pasteur Institute and Iranian Transfusion Organization (unpublished data). * 1US $ = 8300 Iranian Rials ==== Refs Margolis HS Coleman PJ Brown RE Mast EE Sheingold SH Arevalo JA Prevention of Hepatitis B virus transmission by immunization: an economic analysis of current recommendations. JAMA 1995 274 1201 8 7563509 10.1001/jama.274.15.1201 Harbour C Farzadegan H Sharma MK Ala F Professional and voluntary blood: a preliminary study in Iran. Vox sang 1978 34 87 91 622822 Farzadegan H Shamszad M Noori-Arya K Epidemiology of viral hepatitis among Iranian population: a viral marker study. Ann Acad Med Singapore 1980 9 144 8 7425524 Farzadegan H Harbour C Ala F The prevalence of hepatitis B surface antigen and its antibody in blood donors and high-risk groups in Iran. Vox sang 1979 37 182 6 494588 Amini S Mahmoodi MF Andalibi S Solati AA Seroepidemiology of hepatitis B, delta and human immunodeficiency virus infections in Hamadan province, Iran: a population based study. J Trop Med Hyg 1993 96 277 87 8411302 Farhat A Khademi G Mazouman SJ The prevalence of hepatitis B carrier state in Khorassan province of Iran. Saudi Medical Journal 2003 24 549 551 12847639 Bagheri Lankarani K Borhanmanesh F Fattahi F Malekzadeh R Nabipoor I Saberi-Firoozi M Sarafrazyazdi M Reassessment of the role of hepatitis B and C viruses in postnecrotic cirrhosis and chronic hepatitis in southern Iran. Irn J Med Sci 1999 24 117 21 Zamir D Zamir C Rishpon S Epidemiology of hepatitis B virus infection among family members of chronic carriers in Israel. Isr Med Assoc J 2001 3 338 40 11411197 Judson FN Epidemiology of sexually transmitted hepatitis B infections in heterosexuals: a review. Sex Transm Dis 1981 8 336 43 6895799 Aoki H Inaba N Ohkawa R Kudoh J Takamizawa H A study on the possible transmission of hepatitis B virus from HBsAg carrier women to their husbands. Acta Obstet Gynaecol Jpn 1981 33 767 76 7246064 Hu Z Observation on prevention of hepatitis B virus transmission between newly married couples by HBsAg vaccine. Zhonghua Liu Xing Bing Xue Za Zhi 1991 12 222 5 1934045 Zimmerman RK Ruben FL Ahwesh ER Hepatitis B virus infection, hepatitis B vaccine, and hepatitis B immune globulin. J Fam Pract 1997 45 295 315 quiz 317-8 9343051 Keating GM Noble S Recombinant hepatitis B vaccine (Engerix-B): a review of its immunogenicity and protective efficacy against hepatitis B. Drugs 2003 63 1021 51 12699402 Winsnes R Siebke JC Efficacy of post-exposure prophylaxis with hepatitis B immunoglobulin in Norway. J Infect 1986 12 11 21 3958503 Assateerawatt A Tanphaichitr VS Suvatte V Yodthong S Immunogenicity and efficacy of a recombinant DNA hepatitis B vaccine, GenHevac B Pasteur in high risk neonates, school children and healthy adults. Asian Pac J Allergy Immunol 1993 11 85 91 8216564 Palmovic D Crnjakovic-Palmovic J Prevention of hepatitis B virus (HBV) infection in health-care workers after accidental exposure: a comparison of two prophylactic schedules. Infection 1993 21 42 5 8449581 Kimura T Iwamiya M Yoshida N Iwamoto A Sasada M Ito S Kikuchi N Yagi S Higaki M Hepatitis B virus and epidemiological study on efficacy of HBIG and vaccine for the prevention of type B hepatitis after accidental exposures among medical staffs. Kanagawa Shigaku 1989 24 393 8 2489657 Palmovic D Prevention of hepatitis B infection in health care workers after accidental exposure. J Infect 1987 15 221 4 2961815 Type B hepatitis after needle-stick exposure: prevention withhepatitis B immune globulin. Final report of the Veterans Administration Cooperative Study. Ann Intern Med 1978 88 285 93 343678 Update: barrier protection against HIV infection and other sexually transmitted diseases. MMWR Morb Mortal Wkly Rep 42 589 597 1993 Aug 6 8336689 Barnhart KT Sondheimer SJ Contraception choice and sexually transmitted disease. Curr Opin Obstet Gynecol 1993 5 823 8 8286696 Kettering J Efficacy of thermoplastic elastomer and latex condoms as viral barriers. Contraception 1993 47 559 67 8392926 10.1016/0010-7824(93)90023-Z Stone KM Avoiding sexually transmitted diseases. Obstet Gynecol Clin North Am 1990 17 789 99 2092242 Pisu M Meltzer I Lyerla R Cost-effectiveness of hepatitis B vaccination of prison inmates. Vaccine 2002 21 312 21 12450707 10.1016/S0264-410X(02)00457-7 Levaux HP Schonfeld WH Pellissier JM Cassidy WM Sheriff SK Fitzsimon C Economic Evaluation of 2-Dose Hepatitis B Vaccination Regimen for Adolescents. Pediatrics 2001 108 317 25 11483794 10.1542/peds.108.2.317 Lok AS Rose BD Lamivudine monotherapy for chronic hepatitis B infection. UpToDate 2003 UpToDate, Wellesley, MA

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==== Front BMC Infect DisBMC Infectious Diseases1471-2334BioMed Central London 1471-2334-4-321534742910.1186/1471-2334-4-32Research ArticleMutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003 Vega Vinsensius B [email protected] Yijun [email protected] Jianjun [email protected] Wah Heng [email protected] Chia Lin [email protected] Su Yun [email protected] Kin Fai [email protected] Tao [email protected] Prasanna R [email protected] Eng Eong [email protected] Ai Ee [email protected] Lawrence W [email protected] Philip M [email protected] Edison T [email protected] Genome Institute of Singapore, 60 Biopolis Street, Singapore 1386722 Virology Section, Department of Pathology, Singapore General Hospital, Singapore3 Environmental Health Institute, 41 Science Park Road, Singapore Science Park II, Singapore 1176104 Center for Computational Learning Systems, Columbia University, New York, NY 10027 USA2004 6 9 2004 4 32 32 18 5 2004 6 9 2004 Copyright © 2004 Vega et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. Methods We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. Results Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. Conclusions Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs. ==== Body Background The Severe Acute Respiratory Syndrome (SARS) was first reported in November 2002 and rapidly spread to a number of distant global regions by early 2003. A new coronavirus, the SARS-CoV, was identified to be the cause of SARS [1,2] and was rapidly sequenced and characterized [3,4]. SARS-CoV is an enveloped, positive strand RNA virus with a wide host range. Recombination and mutation rates of RNA viruses are high, several orders of magnitude higher than DNA based microbes and in eukaryotes, and have been the cause of rapid changes in antigenicity, virulence, and drug sensitivity. Thus, the direct estimate of the mutation rates of the SARS-CoV in human populations and the analysis of the mutational spectrum would aid in developing strategies for monitoring and therapy. Previously, our analysis of 14 SARS sequences (five of which originated from Singapore) in May 2003 indicated that there are two different genotypes circulating in the world [5]. Recently, there has been a substantial increase in the number of SARS-CoV genomes sequenced. A total of 54 SARS-CoV genomic sequences (37 from the public database prior to October 14, 2003 and 17 sequenced within our institute) are used in our current analysis. This large dataset coupled with the availability of clinical data for cases related to Singapore patients and our molecular observations during in vitro cell passage presents an opportunity for a comprehensive analysis of the SARS-CoV mutational behavior. Methods Viral RNA genome isolation and sequencing SARS-CoV from the primary patient tissues were isolated by homogenizing the tissues in PBS buffer followed by a low speed centrifugation to obtain the viral particle containing supernatant. The virus-containing samples were also inoculated into Vero cell E6. The cells were maintained at 37°C using the usual viral cell culture media, and repassaged after 7 days of incubation. The virus-containing supernatants of homogenize or different passages of Vero cell E6 showing CPE were centrifuged at 23,000 RCF for 2.5 hours to pellet the viral particles and followed by RNA extraction using the QiAmp viral RNA mini kit (Qiagen, ). The RNA genome templates were converted into double strand cDNA and sequenced as previously described [5]. The processing of raw sequence reads (base calling, assembly, and editing) was done using PHRED/PHRAP/CONSED (University of Washington, Seattle, WA, USA, ). Genotype determination using MassArray technology A number of single nucleotide variations (SNVs) were further confirmed using a sensitive Mass Spectrometry based genotyping assay that was developed within our institute [6]. The RNA of the virus was first isolated using QiAmp viral RNA mini kit and then reverse-transcribed into cDNA (using the RNA as template, SuperScript kit from Invitrogen, and sequence specific primers), which were further purified. Primer extension assays were carried out for the SNVs of interest. The extension products were then detected in the MassARRAY (from Sequenom) to determine the genotypes. Data and statistical analysis We aligned the 54 SARS-CoV genomes using CLUSTALW [7]. To minimize the effect of sequencing errors and other artefacts to our analysis, we employed a filtering scheme where only SNVs shared by more than two different isolates are kept. The phylogenetic trees were reconstructed using the filtered variations. The reconstruction was done using PAUP* [8] with Maximum Likelihood criterion, keeping the other parameters to the default. The significance of the variations that pass the proposed mutation filter (where only mutations shared by more than 2 out of 54 isolates are considered real) can be assessed by calculating the probability that a random noise would meet the filtering criterion. The null hypothesis is that the noisy variations are generated independently between genomes. Let q be the rate of noisy mutation in a genome (based on our findings, as reported earlier in the text, we conservatively set , i.e. about two per SARS-CoV genome). The probability that, at a given nucleotide, a noisy mutation is shared by exactly i out of m different isolates is . Thus, the probability that a given nucleotide position has an erroneous mutation shared by more than k isolates is . In a genome with n bases, applying the Bonferroni inequalities, the probability that at least one position is corrupted by noise more than k times is p(k,n,m) ≤ n × s(k,m). In the case of 54 SARS-CoV genomes analyzed in this paper, , m = 54, k = 2, n = 30000, and hence the p-value of mutations that satisfy the filter is ≤ 2.2 × 10-4. In the estimation of SARS-CoV daily mutation rate, we employed the conservative constant mutation rate model [9], where the number of mutations d found in an isolate from its ancestor is proportional to the mutation rate k and the temporal difference t between the isolate and its ancestor, so that d = k × t. Based on the Singapore patients contact tracing information (see Figure 4), we obtained 6 pairs of isolates with known definite ancestor-descendant relationship, calculated the number of mutations (d) and the time difference (t) for each pair, and estimate the mutation rate k for the model using least square fitting. The goodness-of-fit were measured using the adjusted R-square statistics. Another pertinent question in the analysis of SARS-CoV evolution is prediction of the possible date of origin of the human SARS-CoV. Based on the animal-origin hypothesis of SARS-CoV, we assumed the SARS-CoV isolated from palm civet cat as the putative principal isolate that infected the human population. Adhering to the constant mutation rate model, we fit the following model: dx = d0 + kx, where k is the daily rate of mutation, x is the sampling date measured relative to 1st November 2002, and dx is the number of mutations, as compared to the civet cat isolate, of the isolate sampled at date x. Twelve data points were calculated and used to fit the model. The date of origin can be solved by solving x for dx = 0. The goodness-of-fit was measured using the adjusted R-square statistic. Results and discussion SARS-CoV mutations in vitro First, we sought to determine the rate of mutation of the SARS-CoV in Vero cell culture. To achieve this, we subjected SIN2774 isolate to 5 passages in Vero cells. At the appearance of cytolysis at each passage samples were withdrawn and their genomes completely sequenced. Any ambiguities by capillary sequencing were clarified by mass spectroscopic validation [6]. Our results showed that the Vero cell passages are actually comprised of two coexisting quasispecies bearing either an A or G at position 18372. No mutations emerged upon passage, and the ratio of A/G at 18372 remained constant over the passages (Table 1). This suggests that the mutation rate in culture of SARS-CoV is very low at <1 in 5 passages. Thus artificial mutations from limited in vitro cell culture are negligible. Mutations associated with human-to-Vero and Vero-to-human transition Next, we asked whether the transition from human tissue to growth in Vero cell culture engendered either mutations or clonal selection. The SARS-CoV were sequenced from three human tissue-Vero cell culture pairs of viral samples from Singapore and one pair was obtained from the public domain (see supplemental information, Table S1). The results shown in Table 2 showed that mutations emerged in only one case of human to Vero cell passage posted in Genbank (AS → HSR1) but in none of the Singapore pairs. However, the viral sequence from SIN3725V isolated from a lung sample showed evidence for co-infection by two distinct genotypes of SARS CoV. This was manifested by the simultaneous presence of T and C at positions 548, 1727, 13347, confirmed by genotyping using MALDI-TOF MassARRAY technology. Subsequent deconvolution by tracing the "haplotypes" at these loci in different Singaporean isolates revealed probable sequence signatures of T, T, C at these locations in one isolate and C, C, T in the other (see Table S2). Though tissue-derived SIN3725V has two SARS-CoV quasispecies, the isolate after subsequent Vero cell culture showed only one (bearing the T, T, C haplotype, supplemental information, Table S2). These results again show that coinfection by multiple quasispecies is not uncommon in human tissues, and that passage to Vero cells may either generate new mutations at a low rate, or titrates out one quasispecies in the transition. Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker [10]. We sequenced both the originating laboratory isolate (SIN_WNV; see Table S1) and the viral sample directly from the patient's sputum (SIN0409; see Table S2) and found no sequence difference between the two viruses. This reconfirms that the mutation rate from a single point source of virus has a low mutation rate when expanded during human infection. Sequence variation filter Inferring phylogenetic relationships between the known SARS-CoV isolates using existing public data has been problematic because of the potential for sequencing errors. Moreover, the rate of SARS-CoV mutation in culture was not previously known and was thought to be significant given the mutation rates in other RNA viruses. Our experiments provided information as to the potential causes and rates of sequence variations of the SARS-CoV in culture. Based on our Vero cells passages and human-to-Vero transition data, we estimated that, at most, one sequence variation from the original tissue virus can be accounted for by in vitro culture artifacts. The average base-calling error probability (as reported by PHREP [11]) of our sequences is about 7.5 × 10-5, or 2.25 errors per SARS-CoV genome. Accordingly, we suspected that sequencing errors in the reported SARS sequences would be approximately 1–2 bases per reported genome. We used this information to assess the true sequence variants reported in the public SARS sequence databases employing a "mutation filter" [5]. This mutation filter identifies a sequence variant as a probable mutation if it appeared in more than one isolate. Higher filter stringency can be applied by demanding a sequence variant to be present in two, three, or more isolates. A total of 54 isolates were analyzed, including 22 from Singapore. Our results show that the number of mutations appearing in only one isolate is high at 349 (see supplementary Table S3); however, those mutations present in more than two isolates are much lower and appeared relatively stable (Figure 1). Statistical analysis confirmed that the probability of finding any false mutation shared by more than two isolates out of 54 is very low (p ≤ 2.2e - 4) as compared to the probability of finding a false mutation shared by more than one isolate (p ≤ 0.19). These results are consistent with our error estimates as outlined above. We tested the biological validity of this approach by examining the mutational frequency of known genes in the SARS genome. Because of the importance of the 3CL protease and the polymerase for viral replication, we suspected that true non-synonymous mutations in the SARS-CoV present in clinical samples might be rare in these two ORFs in comparison to other structural genes such as those encoding the S, M, and N proteins. Without a mutation filter, sequence variations are commonly observed in the 3CL protease and the polymerase genes. However, when mutations are identified only as variants seen in two or more isolates, then no mutations are detected in the critical 3CL protease and polymerase genes, whereas mutations are noted in the S, M, and N genes regardless of the filter stringency (Figure 2). Therefore, we determined that the most effective mutation filter is presence of a sequence variant in more than two isolates. Molecular history of the viral isolates Using this filter stringency, we assessed the phylogenetic relationship between all 54 isolates describing the recent SARS epidemic. TOR2, Urbani, SIN2500, HKU-39849, CUHK-Su10 formed the core of the early isolates. Employing these as "root", four major clusters appeared: two Singaporean branches, one Taiwan branch, and one North China branch (Figure 3). Validating these clusters, the two sequences from Germany, 'Frankfurt' and FRA, which grouped with the Singaporean branch, were actually derived from the Singaporean doctor who treated the patient SIN2774 and was later hospitalized in Frankfurt. Of the Singaporean cases, SIN2500, SIN2677, SIN2748, and SIN2774 formed one molecular sub-branch which matched with the contact tracing (Figure 4). The clinical contact tracing data was ambiguous as to the direct source of SIN2679's exposure. Intriguingly, however, SIN2679 was the root of a second sub-branch within Singapore that had its origins most probably from the Hotel M cluster. This suggested a potential direct infection of SIN2679 from a Hotel M source other than SIN2500. Using the same contact tracing information, we calculated the average mutation rate during human transmission to be about two mutations per human transmission. Estimation of the mutation rate of the SARS-CoV We obtained the precise dates of symptom onset of 13 Singaporean cases (Table S1). Using the common mutations identified through application of the mutation filter, we employed the constant mutation rate model and estimated the mutation rate of the SARS-CoV during this recent epidemic. We estimated the mutation rate to be 0.1722 nucleotides per day, or 5.7 × 10-6 nucleotide substitutions per site per day (adjusted R-square value of the fitted model = 0.4014). The rates for synonymous and non-synonymous mutations were equivalent at 2.5 × 10-6 and 3.2 × 10-6 nucleotide substitutions per site per day respectively. Using the Singapore isolates with known date of onset, and using the SZ3 and SZ16 genomes isolated from palm civet cat [12] as the putative "original" SARS-CoV that jumped from animal to human, we calculated the daily substitution rate to be 0.1303 nucleotides per day, or 4.3 × 10-6 nucleotide substitutions per site per day, (adjusted R-square = 0.5880) and the estimated possible "date" of SZ3/SZ16 emergence was Oct 21, 2002. Overall, the mutation rate of SARS-CoV appears to be consistent with the reported rate of other viruses [13,14]. The mutational analysis also revealed 5 separate deletions and one insertion that distinguished the different isolates (Figure 5). Intriguingly, they all clustered within a short 200 bp region in the 3' end of the viral genome spanning putative ORFs sars 7b to sars 8b. Despite the overlapping nature of some of the deletions, there was no descendent relationship amongst them and the addition of the insertion/deletion information did not add to the clustering. Our assumption therefore is that this is a region of relative instability that is dispensable for viral replication. Conclusions The focus of this investigation was to measure the mutational frequency and dynamics both in vitro and in vivo of the SARS-CoV. Our findings suggest that the overall SARS-CoV's rate of mutation in culture is low. Inoculation of Human SARS-CoV into Vero cell introduces, on the average, less than one nucleotide mutation. Subsequent culturing of SARS-CoV infected Vero cells induced less than one nucleotide mutation in the five consecutive Vero cell passages. No mutations were also observed during the infection of SARS-CoV cultured in Vero cell to human. This would be consistent with the notion that the SARS-CoV isolates from the patients that gave rise to the in vitro lines are well adapted for in vitro growth. Our proposed mutation filter, which is based on these observations, seems to be stable and effective. Using this filter, reconstruction of molecular phylogenetic relations of the 54 SARS-CoV genomes revealed at least three major branches composed of cases related to Hotel M (Hong Kong), cases reported in North China, and cases found in Taiwan. Moreover, we show that these molecular sequence associations can be effectively used to more precisely reconstruct contact tracing. Our estimated the daily substitution rate of SARS-CoV to be 0.1722 nucleotides, or 5.7 × 10-6 nucleotide substitutions per site, a mutation rate similar to other RNA viruses [13,14]. Taking the SARS-CoV isolated from palm civet cat as the putative originating SARS-CoV, our calculations suggest that the earliest possible date for SARS emergence is predicted to be Oct 21, 2002. During the final preparation of this manuscript two reports were published addressing the mutation rate of the SARS-CoV in human populations. Yeh et. al. [15], examining Taiwanese SARS samples estimated the CoV mutation rate to be about 1.83 × 10-6 nucleotides per site per day. The Chinese SARS Molecular Epidemiology Consortium [16], examining a larger number of viral isolates, recently determined the mutation rate to be 8.26 × 10-6 nucleotides per site per day using samples from China. These estimates were very close to ours. In addition, The Chinese Consortium [16] projected the time of emergence of the SARS CoV epidemic to be November 2002. The remarkable consensus of these three studies using different patient populations on the mutational dynamics of the SARS CoV suggests that these results are bona fide. Competing interests None declared. Authors' contributions VBV and ETL performed most of the data analysis and prepared the draft manuscript. STSY and LAE provided SARS-CoV samples from patients and Vero cells. KFT and OEE provided continue SARS-CoV containing Vero cell passage samples. YR and LWS designed the experiments and coordinated sample acquiring and the viral genome sequencing. CLW and TZ generated all DNA sequence data. WHL processed all DNA sequence and assembled the viral genomes. JL verified all sequence variations by conducting MALDI-TOF mass spectrometry analysis. PK assessed the effects of nucleotide variations to ORFs and their proteins. VBV and PML carried out the mathematical and statistical analysis. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Table S1 List of the sequences used in the analysis. This list is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file Additional File 2 Table S2 Single nucleotide variations (SNVs) detected initially in capillary sequencing (second row) and subsequently confirmed by MALDI-TOF MS-based genotyping (first row). This table is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file Additional File 3 Table S3 Complete list of single nucleotide variations (SNVs) observed in the 54 SARS-CoV isolates. The Singapore sequences used were all based on capillary sequencing. This data is available at http://giscompute.gis.a-star.edu.sg/sars_mut_dyn/ Click here for file Acknowledgements We want to express their appreciation to Mr. Thoreau Herve, Mr. Landri Lim, Ms. Carine Bonnard, Mr. Meah Wee Yang, and Ms. Lin Su for providing technical support, and Mr. Chia Jer Ming for assisting the ORF analysis. This study was supported by the Agency for Science, Technology, and Research of Singapore, and the Biomedical Research Council of Singapore. The authors wish to express their appreciation to Mr. Thoreau Herve, Mr. Landri Lim, Ms. Carine Bonnard, Mr. Meah Wee Yang, and Ms. Lin Su for providing technical support, and Mr. Chia Jer Ming for assisting the ORF analysis. This study was supported by the Agency for Science, Technology, and Research of Singapore, and the Biomedical Research Council of Singapore. Figures and Tables Figure 1 Effects of applying mutation filter to number of substitutions observed. To exclude nucleotide variations arising from sequencing error or other artifacts, we include only variations that are present in at least a number of different isolates. The graph shows an exponential decrease in the number of nucleotide variations detected with increasing filter stringency. The curve essentially flattens around 3, which corresponds to the filtering scheme that considers a sequence variation as a real mutation if it is shared by more than two isolates. Figure 2 Non-silent variations in five key ORFs. Nucleotide variations that lie within a coding region might result in amino acid variations in the corresponding protein product. Such non-silent variations could play a significant role in determining the survivability of SARS-CoV variants. Application of the proposed variation filter reveals conservation of the polymerase (RdRp) and 3CLpro, which is consistent with previous studies of other Corona viruses. Figure 3 Molecular relationship between 54 SARS-CoV genomes. The phylogenetic tree reconstructed using PAUP* on nucleotide variations shared by more than two isolates. The tree was re-rooted on the earliest reported case, i.e. TOR2. Four major branches can be observed in the tree, each largely belonging to a certain geographical origin. Two of which encompass the Singapore cases, indicating the possibility of separate infection sources of the Singapore cases. Figure 4 Relations between the Singapore patients and others related to Hotel M, Hong Kong. Contact tracing information of the Singapore patients and the two early reported SARS cases. Contact information revealed a single source of infection for Singapore patients, but molecular analysis, reported here and in (6), suggests the existence of an additional infection source (represented by dashed arrows and box) from the Hotel M case to Singapore. Figure 5 Map of significant mutations, insertions and deletions in the SARS-CoV genome. Map of the SARS genome, plotted with substitutions appearing in more than two isolates and other major insertion and deletion regions. Golden bars signify the SARS-CoV genome, with the approximate nucleotide positions shown in the scale on top of it. Arrows drawn on top of the genome indicate nucleotide mutations (i.e. variations shared by more than two isolates) observed in the SARS-CoV genome. Amino acid changes in SARS-CoV's proteins are reflected as arrows on top of the protein bars (blue bars). Significant multiple-nucleotide deletions (pink bar) and insertions (light-blue bar) were also observed (denoted as arrows under the genome) and appear to cluster around position 27000nt to 28000nt. Table 1 Table 1: Single nucleotide heterogeneity (SNH) observed in the six passages of Vero cells culture. Along the six Vero cell passages, nucleotide heterogeneity was observed (initially through capillary sequencing and confirmed using MassARRAY genotyping) at nucleotide position 18356. Presence of single nucleotide heterogeneities (SNHs) indicates coexistence of multiple SARS-CoV isolate in the Vero cell culture. Genomic Location (Based on SIN2774) SIN2774_P1 SIN2774_P2 SIN2774_P3 SIN2774_P4 SIN2774_P5 ORF 18356 A/G A/G A/G A/G A/G 135aa of nuclease ExoN homolog [R/G] Table 2 Quasispecies fluctuations and mutations during the transition from human tissue to Vero cell culture. Nucleotide variations observed between primary human tissue isolates and their respective subsequent Vero cell culture. Both quasispecies selection (SIN3275V → SIN849M) and new emergence (AS → HSR1) are observed during the transmission of SARS-CoV from human tissue sample into Vero cell culture. Source of viral sequence: Human Tissue Source of viral sequence: Passage to Vero cell culture Nucleotide Heterogeneity Position (Urbani) ORF (based on NC_004718.3) Sin3408L Sin842M - - - Sin3725V Sin849M Y [t/C] → T 548 95aa of Leader protein (I, T → I) Y [T/C] → C 13347 131aa of NSP10 (SILENT) Sin3765V Sin852M - - - AS HSR 1 G → R [G/A] 27254 637aa of sars6 (D → D, N) Table 3 Nucleotide variations during the transition from Vero cell culture to human. No nucleotide variations were observed between isolate from laboratory-acquired SARS patient and its infection source. Source of viral sequence: Vero cell culture Source of viral sequence: Passage to Human Tissue Number of stable nucleotide substitutions SinWNV Sin0409 (sputum) 0 ==== Refs Ksiazek TG Erdman D Goldsmith CS Zaki SR Peret T Emery S Tong S Urbani C Comer JA Lim W Rollin PE Dowell SF Ling AE Humphrey CD Shieh WJ Guarner J Paddock CD Rota P Fields B DeRisi J Yang JY Cox N Hughes JM LeDuc JW Bellini WJ Anderson LJ SARS Working Group A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome New England Journal of Medicine 2003 348 1953 1966 12690092 10.1056/NEJMoa030781 Drosten C Gunther S Preiser W van der Werf S Brodt HR Becker S Rabenau H Panning M Kolesnikova L Fouchier RA Berger A Burguiere AM Cinatl J Eickmann M Escriou N Grywna K Kramme S Manuguerra JC Muller S Rickerts V Sturmer M Vieth S Klenk HD Osterhaus AD Schmitz H Doerr HW Identification of a Novel Coronavirus in Patients with Severe Acute Respiratory Syndrome New England Journal of Medicine 2003 348 1967 1976 12690091 10.1056/NEJMoa030747 Marra MA Jones SJ Astell CR Holt RA Brooks-Wilson A Butterfield YS Khattra J Asano JK Barber SA Chan SY Cloutier A Coughlin SM Freeman D Girn N Griffith OL Leach SR Mayo M McDonald H Montgomery SB Pandoh PK Petrescu AS Robertson AG Schein JE Siddiqui A Smailus DE Stott JM Yang GS Plummer F Andonov A Artsob H Bastien N Bernard K Booth TF Bowness D Czub M Drebot M Fernando L Flick R Garbutt M Gray M Grolla A Jones S Feldmann H Meyers A Kabani A Li Y Normand S Stroher U Tipples GA Tyler S Vogrig R Ward D Watson B Brunham RC Krajden M Petric M Skowronski DM Upton C Roper RL The Genome Sequence of the SARS-Associated Coronavirus Science 2003 300 1399 1404 12730501 10.1126/science.1085953 Rota PA Oberste MS Monroe SS Nix WA Campagnoli R Icenogle JP Penaranda S Bankamp B Maher K Chen MH Tong S Tamin A Lowe L Frace M DeRisi JL Chen Q Wang D Erdman DD Peret TC Burns C Ksiazek TG Rollin PE Sanchez A Liffick S Holloway B Limor J McCaustland K Olsen-Rasmussen M Fouchier R Gunther S Osterhaus AD Drosten C Pallansch MA Anderson LJ Bellini WJ Characterization of a Novel Coronavirus Associated with Severe Acute Respiratory Syndrome Science 2003 300 1394 1399 12730500 10.1126/science.1085952 Ruan YJ Wei CL Ee AL Vega VB Thoreau H Su ST Chia JM Ng P Chiu KP Lim L Zhang T Peng CK Lin EO Lee NM Yee SL Ng LF Chee RE Stanton LW Long PM Liu ET Comparative Full-length Genome Sequence Analysis of 14 SARS Coronavirus Isolates and Common Mutations Associated with Putative Origins of Infection Lancet 2003 361 1779 1785 12781537 10.1016/S0140-6736(03)13414-9 Liu JJ Lim SL Ruan Y Ling A Drosten C Liu ET Stanton LW Hibberd ML SARS-CoV Transmission Epidemiology Revealed by MALDI-TOF Mass Spectrometry-based Viral Genotyping: 2004 submitted Thompson JD Higgins DG Gibson TJ CLUSTAL-W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-specific Gap Penalties and Weight Matrix Choice Nucleic Acids Research 1994 22 4673 4680 7984417 Swofford DL PAUP: Phylogenetic Analysis Using Parsimony (and Other Methods) Version 4 2003 Sunderland, Massachusetts: Sinauer Associates Nei M Kumar S Molecular Evolution and Phylogenetics 2000 Oxford University Press, Oxford Lim PL Kurup A Gopalakrishna G Chan KP Wong CW Ng LC Se-Thoe SY Oon L Xinlai Bai X Stanton LW Ruan Y Miller LD Vega VB James L Ooi PL Kai CS Olsen SJ Ang B Leo YS Laboratory-acquired Severe Acute Respiratory Syndrome (SARS) – Singapore New England Journal of Medicine 2004 Ewing B Green P Base-calling of automated sequencer traces using phred. II. Error probabilities Genome Research 1998 8 186 194 9521922 Guan Y Zheng BJ He YQ Liu XL Zhuang ZX Cheung CL Luo SW Li PH Zhang LJ Guan YJ Butt KM Wong KL Chan KW Lim W Shortridge KF Yuen KY Peiris JS Poon LL Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Science 2003 302 276 8 12958366 10.1126/science.1087139 Drake JW Holland JJ Mutation Rates Among RNA Viruses PNAS 1999 96 13910 13913 10570172 10.1073/pnas.96.24.13910 Li WH Tanimura M Shrap PM Rates and Dates of Divergence Between AIDS Virus Nucleotide Sequences Molecular Biology and Evolution 1988 5 313 330 3405075 Yeh SH Wang HY Tsai CY Kao CL Yang JY Liu HW Su IJ Tsai SF Chen DS Chen PJ Chen DS Lee YT Teng CM Yang PC Ho HN Chen PJ Chang MF Wang JT Chang SC Kao CL Wang WK Hsiao CH Hsueh PR Characterization of Severe Acute Respiratory Syndrome Coronavirus Genomes in Taiwan: Molecular Epidemiology and Genome Evolution PNAS 2004 101 2542 2547 14983045 10.1073/pnas.0307904100 The Chinese SARS Molecular Epidemiology Consortium Molecular Evolution of the SARS Coronavirus During the Course of the SARS Epidemic in China ScienceExpress 2004 10.1126

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==== Front BMC Med ImagingBMC Medical Imaging1471-2342BioMed Central London 1471-2342-4-31532915210.1186/1471-2342-4-3Research ArticleHistologic assessment of biliary obstruction with different percutaneous endoluminal techniques Rossi Michele [email protected] Vito [email protected] Filippo Maria [email protected] Alberto [email protected] Laura [email protected] Luigi [email protected] Giampiero [email protected] Elisa [email protected] Vincenzo [email protected] Department of Radiology, "S. Andrea" Hospital-II Faculty "La Sapienza" University, Rome,00100, Italy2 Department of Radiology, "UmbertoI" Hospital-I Faculty "La Sapienza" University, Rome,00100, Italy3 Department of Radiology, "Annunziata Civil Hospital"-Cosenza, 87100, Italy2004 25 8 2004 4 3 3 4 9 2003 25 8 2004 Copyright © 2004 Rossi et al; licensee BioMed Central Ltd.2004Rossi et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Despite the sophisticated cross sectional image techniques currently available, a number of biliary stenosis or obstructions remain of an uncertain nature. In these pathological conditions, an "intrinsic" parietal alteration is the cause of biliary obstruction and it is very difficult to differentiate benign from malignant lesions using cross-sectional imaging procedures alone. We evaluated the efficacy of different endoluminal techniques to achieve a definitive pathological diagnosis in these situations. Methods Eighty patients underwent brushing, and or biopsy of the biliary tree through an existing transhepatic biliary drainage route. A subcoort of 12 patients needed balloon-dilatation of the bile duct and the material covering the balloon surface was also sent for pathological examination (balloon surface sampling). Pathological results were compared with surgical findings or with long-term clinical and instrumental follow-ups. Success rates, sensitivity, specificity, accuracy, confidential intervals, positive predictive value and negative predictive value of the three percutaneous techniques in differentiating benign from malignant disease were assessed. The agreement coefficient of biopsy and brushing with final diagnosis was calculated using the Cohen's "K" value. Results Fifty-six patients had malignant strictures confirmed by surgery, histology, and by clinical follow-ups. Success rates of brushing, balloon surface sampling, and biopsy were 90.7, 100, and 100%, respectively. The comparative efficacy of brushing, balloon-surface sampling, and biopsy resulted as follows: sensitivity of 47.8, 87.5, and 92.1%, respectively; specificity of 100% for all the techniques; accuracy of 69.2, 91.7 and 93.6%, Positive Predictive Value of 100% for all the procedures and Negative Predictive Value of 55, 80, and 75%, respectively. Conclusions Percutaneous endoluminal biopsy is more accurate and sensitive than percutaneous bile duct brushing in the detection of malignant diseases (p < 0.01). Biliary neoplasmsBile ductsbiopsyBile ductscytologyPercutaneus-cholangioscopy ==== Body Background Bile duct dilatation and increasing jaundice are often onset symptoms of a number of either malignant or benign pathological conditions. In most cases, common cross-sectional imaging techniques such as ultrasonography (US) or spiral-CT or cholangio-pancreatico-biliary magnetic resonance (MRCP) are highly capable of depicting the causes of obstruction by simply showing extrinsic masses in case of infiltrating tumors or benign causes of jaundice, like stones [1]. These non invasive techniques, however, can still yield uncertain results in some particular benign or malignant pathological conditions. When stones are too small for example, or when bile duct dilatation is limited. For final confirmation or exclusion of endobiliary stones ERCP is unavoidable before the subsequent therapeutic steps. There are also some pathological conditions where an "intrinsic" parietal alteration is the cause of biliary obstruction and it is very difficult to distinguish benign from malignant lesions using cross-sectional imaging procedures alone (figure 4 - 5). A biliary dilatation may also occur after surgical interventions involving the biliary system, like bilioenteric anastomoses for gastric or pancreatic neoplasms or cholecystectomies. It is not always possible to distinguish between a recurrent disease or a fibrotic post-surgical stenosis, especially if it occurs early and if inflammatory alterations were already present during surgery. A number of obstructions remains therefore unexplained because the aforementioned imaging modalities do not show any extrinsic compressing or infiltrating mass, nor a calcolous disease. In these cases, only the intrahepatic bile ducts dilation and the level of the obstruction can be determined, but additional diagnostic procedures should be performed for a diagnosis of the real nature of the obstruction [2]. Patients with obstructive jaundice often undergo percutaneous biliary cholangiography and drainage (PBD) for decompression of the biliary tree. Although relatively invasive, not even PBD allows to achieve a definitive differential diagnosis between malignant and benign pathologies. Tissue sampling becomes therefore essential for the histological characterization of parietal alterations and for planning their appropriate treatment [3]. Bile cytology, although easily feasible either through a transhepatic or an endoscopic route, does not accomplish adequate pathological diagnoses in most cases [5-7]. In our study, three different endoluminal sampling techniques for the characterization of biliary strictures of uncertain nature have been reviewed and comparatively evaluated with the purpose of determining which one of them is the most accurate for differentiating between benign or malignant pathology. Methods Our investigation was performed on a population of 80 patients who, from January 1992 to September 1999, underwent endoluminal brushing and/or biopsy for biliary stenosis or obstructions of uncertain nature. A retrospective evaluation of the efficacy of these techniques was carried out by comparing the pathological findings on one hand and radiological findings, clinical long-term follow ups, and/or post-surgical specimens, on the other. Patient population included 43 men and 37 women, with a mean age of 62 years (range 37–87 years). All patients presented jaundice as a common symptom, weight loss was present in 20 cases (25%), and itching in 12 cases (15%). At admission, all patients presented biliary dilation, but no masses were identifiable by imaging procedures such as US, CT, MRCP. ERCP had been performed in 23 patients with middle-low common bile duct stenosis but the nature of obstruction could not be determined nor a drainage could be placed. Obstruction were located at the biliary bifurcation in 18 cases (22.5%), the common extrahepatic bile duct in 50 (62.5%), the right (2) and left (2) hepatic duct in 4 cases (5%) Eight patients with bilioenteric anastomoses (10%) were also evaluated in order to determine the nature of the stricture. The patients came to the interventional unit needing for a biliary drainage. PBDs were performed using standard interventional techniques.[5] The percutaneous approach was right lateral in all patients but 25 of them required also an anterior subxyphoid approach. The procedures for cytological and/or histological samples were performed in any case 3–5 days after placement of the biliary drainage in order to operate on a decompressed biliary system. One hundred and two endoluminal procedures were included in this study. Twenty-one patients underwent cytologic brushing only, 25 endoluminal biopsy only, and 22 both brushing and biopsy. A total number of 43 brushings and 47 biopsies were performed. In 12 additional cases, we obtained cytological and tissue samples from an angioplastic balloon used for the bilioplasty of strictures of uncertain nature (balloon surface sampling) with techniques that will be described. Sixteen patients underwent brushing only, because the cholangioscope was not available yet and five because the diagnosis of malignancy achieved with brushing was considered sufficient, thus not requiring further diagnostic workups to planning the appropriate therapy. All the specimens were collected after PBD thus after translesional advancement of a 10 French catheter; in 12 particularly heavy stenosis a preliminary balloon dilatation was also accomplished. Brushing was performed according to the following technical steps: - the indwelling biliary drainage catheter was exchanged over a guide-wire with a 7/9-F. introducer sheath without valves at the proximal end ("peel-away", William Cook, Europe) - a flexible probe (Fig. 3) with a cilindrical brush at the tip, 5 mm in diameter 10 mm in length (Olympus Italia srl. Code number BC20295010), was advanced through the sheath until it was beyond the lesion; the sheath was withdrawn to expose the brush, which was then pulled back and forth and rotated across the lesion several times under fluoroscopic control Figure 3 A metallic flexible probe with a cilindrical brush at the atraumatic tip, 5 mm in diameter 10 mm in length(Olympus Italia srl. Code number BC2029501) brush (approximately 1 cm in length and 5 mm in diameter) - the brush was then carefully pulled back to be removed from the patient into the sheath, to avoid malignant spreading through the transhepatic tract -samples were then placed on a glass slide, fixed with Sprayfix (Surgipath Medical Ind.; Illinois, USA), immediately submitted for cytological examination and stained by the standard Papanicolau technique. Endoluminal forceps biopsy was performed under direct visualization with a 5-mm. cholangioscope (Olympus URF. Type P, Japan) (Fig. 1). Sometimes, the site of the lesion endoscopically visible did not correspond to the site of the stenosis fluoroscopically detectable; additional samples were therefore collected under fluoroscopic guidance, according to the following technique: Figure 1 Flexible cholangioscope (5 mm in diameter) (Olympus, URF, type P, Japan) - the biliary catheter was replaced, over a guide, by a 18-F "peel-away" introducer sheath through which the cholangioscope was advanced until it was close to the lesion; - a flexible forceps (5-F. Olympus, FB 185X Fig. 2) was passed through the working channel of the cholangioscope to obtain 3–4 specimens for each patient; Figure 2 Alligator forceps (Olympus, FB 195X, Japan). The wire tip is open 25 - the specimens were fixed in a 10%-solution of formaline and sent to the pathologist, included in paraffin, and dissected in slices of 2–4 microns at microtome; subsequently, they were stained by hematoxilyn-eosin, PAS and Mallory techniques; - after completion of the biopsy procedures, all the patients underwent cholangiographic control checking for contrast material extravasation at the biopsy site and monitored for symptoms of hemobilia and/or bacteriemia. All the patients were submitted to antiobiotic therapy before and during the three days following endobiliary procedures. Twelve patients underwent preliminary bilioplasty because of the difficulties in advancing the drainage catheter across the biliary stricture. In this group, a different sampling was performed, based on the examination of those cells and/or tissue remained stuck to the angioplastic balloon after dilatation (balloon surface sampling): - once deflated, the balloon (8–10 mm., Meditech, Boston, USA) was pulled back into the sheath and removed from the patient; - the balloon was then placed in a saline solution with 10% of formalin, inflated and agitated several times to facilitate detachment of samples, which were then immediately delivered to the pathology laboratory. Pathological specimens from 14 patients were compared with open surgery findings and post-surgical pathological reports. The pathologists, hystologists and cytologists, were blinded as to corresponding results. The follow-up of the patients undergone to surgery stopped after the operation. Patients not candidate to surgery were treated with interventional procedures only such as bilioplasty, biliary drainage, interstitial radiotherapy and/or with chemotherapy or just a supportive therapy. This group of unoperated patients was followed-up on the basis of clinical and radiological data obtained by case-record reviews and by correspondence with their referring clinicians and general practitioners. The follow-up period ranged from 3 to 48 months. A minimum of 12 months of healthy negative cytological-histological diagnosis was necessary because either a clinically benign stenosing lesion or a cytohistological diagnosis negative for malignancy must be confirmed by a prolonged survival, as well as by clinical and radiological findings. Calculations of success rate, sensitivity, specificity, positive predictive value, and negative predictive value for each technique were based on the number of biopsy procedures (n = 102) rather than on the number of patients (n = 80), (Table 1) since each biopsy was considered as a separate event The success rate is the percentage of biopsy procedures resulting in sufficient material for microscopic evaluation. Table 1 Differentiation of biliary obstruction with different percutaneous endoluminal techniques Technique N. PTS SR TP TN FP FN Brushing 43 * 39/43 11 16 0 12 Biopsy 47 * 47/47 35 9 0 3 Balloon Brushing 12 12/12 7 4 0 1 SR= Success Rate; TP= True Positive; TN= True Negative; FP= false positive; FN= False Negative *22 patients underwent either brushing and forceps biopsy. Confidential Intervals (CIs) were determined for brushing and biopsy groups assuming a P value of .01 by using the Geigy Scientific Table (Geigy, Florence, 1984). The agreement coefficient between biopsy or brushing and final diagnosis was calculated using the Cohen's "K" value using SPSS 8.0 for Windows (SPSS, Chicago, Illinois, 1997). Results and discussion A final diagnosis of malignant disease was confirmed in 56 (70%) cases and a final diagnosis of benign disease in 24 (30%) cases for an overall of 80 patients. Final diagnoses in the malignant group included: cholangiocarcinoma (n = 31), adenocarcinoma (n = 6), metastatic adenocarcinoma (n = 5), pancreatic carcinoma (n = 13) and malignant endocrine tumor (n = 1). Final diagnoses in the benign group included: iatrogenic stenosis (n = 9), sclerosing cholangitis (n = 12) and primary (N = 2) and secondary biliary cirrhosis (N = 1). Thirty-nine of the 43 brushing biopsies procedures were technically adequate for the diagnostic evaluation. Four cases were considered "poorly cellular" by the pathologists, with an overall success rate of 90.7% (Table 1). These 4 cases underwent endoluminal forceps biopsy within 15 days from the brushing. Malignant cells were detected by brushing in 11 cases including: adenocarcinoma (n = 2), cholangiocarcinoma (n = 6), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n= 1) (table 2). Table 2 True positives Type of tumour Brushing Biopsy Balloon-brushing Cholangiocarcinoma 6 18 5 Adenocarcinoma 2 1 1 Metastatic adenoca. 1 5 - Pancreatic carcinoma 2 10 1 Neuroendocrine tumor 1 - Of the 32 patients with negative findings for malignant cells, the 4 cases in whom the samples were considered "acellular" by the pathologists were excluded by the statistical analysis, as just mentioned. Hence, 16 out of 28 had a final diagnosis of benign disease confirmed by long-term clinical follow-up (true negatives) (Table 3) and 12 patients had a final diagnosis of malignancy (false negatives) (Table 4). Therefore, 11 true positives, 16 true negatives and 12 false negatives were obtained by cytological brushing, with an overall sensitivity, specificity, accuracy, PPV, and NPV in the detection of malignant diseases of 47.8, 100, 69.2, 100, and 57.1 % respectively (Table 1,5). Table 3 True negatives Type of tumor Brushing Biopsy Balloon-brushing Sclerosing cholangitis 10 4 - Biliary cirrhosis 3 2 - Iatrogenic strictures 3 3 4 Table 4 False negatives Type of tumor Brushing Biopsy Balloon-brushing Cholangiocarcinoma 7 - 1 Adenocarcinoma 2 1 - Pancreatic carcinoma 3 2 - Table 5 Statistical analysis Technique Sensitivity Specificity Accuracy PPV NPV Brushing 47.8% 28.10–69.66* 100% 87.30–100.00* 69.2% 100% 57.1% Biopsy 92.1% 75.56–98.53* 100% 89.34–100.00* 93.6% 100% 75% Balloon brushing 87.5% 52.30–99.96* 100% 64.31–100.00* 91.7% 100% 80% * 99% C.I. (Geigy scientific tables) Endoluminal forceps biopsy was performed in 47 cases. Thirty-five of these were interpreted as containing malignant cells (Table 2): cholangiocarcinoma (n = 18), pancreatic carcinoma (n = 10), metastatic adenocarcinoma (n = 5), adenocarcinoma (n = 1), and neuroendocrine tumor (n = 1). Nine of the 12 cases interpreted as containing inflammatory cells were confirmed by clinical and radiological follow-up as follows: sclerosing cholangitis (n = 4), iatrogenic stenosis (n = 3) and biliary cirrhosis (n = 2). Three cases were erroneously interpreted as benign (false negative) but the clinical follow-up revealed 2 pancreatic carcinomas and 1 adenocarcinoma (Table 4). With 35 true positives, 9 true negatives and 3 false negatives, endoluminal forceps biopsy showed a sensitivity of 92.1%, a specificity of 100%, an accuracy of 93.6%, and a PPV and NPV of 100 and 75%. CIs were reported in Table 5. "K" values for biopsy and brushing vs clinical/surgical final diagnosis were 0.613 and 0.404, respectively (Table 6). Table 6 Cohen's Kappa value Cohen's Kappa Significance Brushing vs follow-up 0.404 0.001 Biospy vs follow-up 0.613 0.019 As previously discussed, 12 samples were obtained in as many patients with an angioplastic balloon after dilation of biliary strictures (balloon surface sampling). This technique demonstrated malignant cells in 7 (58.3%) cases and benign cells in 5 (41.7%). The final clinical diagnosis of the malignant group included adenocarcinoma (n = 1), cholangiocarcinoma (n = 5) and pancreatic carcinoma (n = 1) (Table 2). The final clinical diagnosis of the benign group included four iatrogenic stenosis (Table 3). In one case a sclerosing cholangitis was diagnosed by balloon brushing, but clinical follow-up and further investigations revealed a cholangiocarcinoma (Table 4). With this technique, we therefore had 1 false negative, 7 true positives and 4 true negatives, with a sensitivity, specificity, accuracy, positive predictive value and negative predictive value of 87.5, 100, 91.7, 100, 80%, respectively (Table 5). Twenty-two patients underwent brush cytology together with endoluminal forceps biopsy. By excluding the 4 patients whose specimens were considered "acellular" and comparing each procedure with the clinical follow-up and surgical specimens, we obtained 12 concordant and 6 discordant diagnoses. In the group of 12 concordant diagnoses, 6 benign diseases, such as primary sclerosing cholangitis (n = 3), iatrogenic stricture (n = 1) and biliary cirrhosis (n = 2) and 6 malignant diseases, such as cholangiocarcinoma (n = 3), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n = 1) were included. All concordant diagnoses were confirmed by clinical or surgical follow-up. In the group of 6 discordant diagnoses, cholangiocarcinoma (n = 4) adenocarcinoma (n = 1) and pancreatic carcinoma (n = 1), were included. All the 6 malignancies misdiagnosed by brushing were correctly diagnosed by biopsy. Transient hemobilia (spontaneously reversed from 1 to 12 hours after the procedure), was observed in 5/47 (10.6%) patients who had undergone biopsy. In 4 of them, particular angulations of the access route was present and there were difficulties in negotiation of the stricture. No major complications directly related to the brush cytology or the endoluminal forceps biopsy procedures occurred. Discussion The evaluation of patients with biliary tract obstruction without evidence of any intrahepatic or extrahepatic growing mass has traditionally involved a variety of diagnostic imaging techniques [1]. Ultrasound and CT are often the initial diagnostic investigation to be performed and provide good information about the presence of biliary obstructions and the degree of ductal dilatation [8]. These imaging modalities, are however limited in depicting intraductal anatomy and, sometimes, in exactly determining the level and the cause of the obstruction [1,9]. Magnetic resonance cholangiography (MRC) is a non-invasive imaging modality providing a good visualization of the biliary. system. The sensitivity of MRC in the detection of choledocholithiasis has been reported as 90–100%, a comparable rate with that of endoscopic retrograde cholangiopancreatography (ERCP) [10-15]. The assessment of the level of obstruction also has been reported as highly accurate [16-19]. However, low-grade strictures or lesions causing biliary dilation may be missed by MRC [20]. The pathological characterization of presumed malignant strictures can be therefore, difficult, if not impossible, using noninvasive imaging studies alone in some intrinsic lesions causing biliary dilation. As a final diagnosis could radically affect further therapeutic choices histological, an histological characterization is required in the management of patients with biliary strictures [1]. Fine-needle percutaneous biopsy (FNPB) and fine-needle aspiration (FNA) has been reported poorly valuable in absence of a lesion clearly identifiable [21,22]. The tumor most frequently not identifiable as a true growing mass is cholangiocarcinoma and the differential diagnosis with primary sclerosing cholangitis is of somewhat importance [23]. The differential diagnosis between some carcinomas of the pancreatic head or small submucosal tumors of the ampulla and cholangiocarcinoma or inflammatory diseases, such as sclerosing cholangitis, can be very difficult in many cases. Patients with bilioenteric anastomoses after tumoral mass resection have a very complex local anatomic alteration that makes extremely difficult any radiological investigation searching for small recurrent neoplastic infiltrations [27]. Treatment protocols require pathological diagnoses for palliative or possibly curative therapy in almost all these types of conditions. In addition, the surgical technique itself can be different if a malignancy is present or not. Some Authors, when liver transplant is still indicated, suggest a large excision associated with gastrectomy, pancreatectomy and a transverse ascending colectomy [25]. Although the efficacy of this surgical approach is still under debate, the role of a preoperative diagnosis is extremely important. Malignant cells surrounding the biliary ducts may continuously exfoliated into the bile, especially in the case of tumors which breack through the mucosa, and become available for the cytological examination when the bile is collected either percutaneously or by ERCP. Cytodiagnosis is easy to be performed, atraumatic in nature, with less potential risk and associated with relatively low charges. This technique has shown low sensitivity (15–28%) and accuracy (48–58%) rates, due to an early cellular degeneration after bile collection and to "poorly cellular" specimens. In addition, some pathological aspects can affect the efficacy of the procedure, such as in case of lesions extrinsically compressing the bile duct wall without a complete transmural infiltration or an adequately wide mucosal disruption [22]. Endoluminal brush cytology was successfully performed either percutaneously, by Radiologists, or endoscopically, by Gastroenterologists [26-28]. In our experience, brush cytology showed a high specificity but low sensitivity and accuracy rate in the detection of malignant diseases. Technical limits were mostly represented by "poorly cellular samples". In our study, in fact, 4 cases (9.3%) were considered "poorly cellular" by the pathologists. In addition to these technical problems, similarly to bile cytology, we have to consider some morphological aspects that can negatively influence the sensitivity of this technique. According also with our cholangioscopic experience, biliary tumors may remain intramural causing an annular constriction of the biliary duct [21,29] without complete transmucosal infiltration, and this condition can mimic sclerosing cholangitis and render brush cytology ineffective. In light of the fact that there is a frequent relationship between sclerosing cholangitis and malignant strictures, the cytological differentiation between inflammatory and malignant changes can be extremely difficult. In addition, these tumors are frequently so well differentiated that their identification as "malignant" can be, even histologically, difficult [29,30]. These are the theoretical explanations that we can give to the fact that cytological diagnosis of cholangiocarcinoma yielded a relatively low sensitivity, verified in our study as well as in the literature [1,21-23,26,30]. At endoscopic sampling, cholangiocarcinoma has a higher sensitivity [28] In this review only patients not suitable for ERCP or coming from a failed drainage or other type of retrograde endoscopic intervention were evaluated, thus the percutaneous and endoscopic techniques can not be compared. Whenever possible especially if skilled endoscopists are available, retrograde approach could still to be considered the first step, for its potentially high sensitivity especially if repeated sampling are performed [27,28] Percutaneous brush cytology, if compared with bile transendoscopic cytodiagnosis, has the potential risk of a malignant spreading of cells through the transhepatic tract. To overcome this risk, in our opinion, the brush should be pulled back and removed from the patient into the introducer sheath. With this technique, in fact, no spreading along the transhepatic tract was observed in our malignant patients at imaging follow-up. Brush cytology is a easy, safe and at relatively low-cost procedure, similarly to bile collection. A single sampling however has a low possibility of detecting a malignancy. The results can improve with multiple samples. Three consecutive negative samples decrease the probability of a malignancy from more than 55% to less than 5% [30]. The absence of false positives in our and others, series [2] means that an intraductal biopsy has no purpose when an exfoliative cytology is positive. Meanwhile, in presence of a negative cytology, other techniques such as percutaneous FNA and endoluminal forceps biopsy should be mandatory. Fine needle aspiration performed either percutaneously or endoscopically has some technical advantages over endoluminal brushing in those lesions extrinsically compressing the biliary duct without deeply infiltrating the ductal wall, since the inner epithelial layer is not involved [21]. Most of these patients with biliary obstruction, especially with high lesions, however, in our Hospital, usually undergoes percutaneous biliary drainage in the early phases of their clinical work-up. A percutaneous FNA would represent an adjunctive interventional procedure that could be avoided by using the transhepatic route, already available. We had adequate percutaneous biopsy specimens in epithelial lesions, such as cholangiocarcinoma, and inadequate specimens in those lesions with inflammatory and/or necrotic changes. The dense fibrotic and scirrus reaction associated with pancreatic carcinoma may result in poor biopsies specimens [31]. In addition, pancreatic carcinoma is often associated with pancreatitis, necrotic cellular debris and a dense fibrotic reaction which can further contribute to the disappointing results obtained with even aggressive percutaneous biopsy techniques [32]. The differentiation degree of a tumor can affect the accuracy of the histological classification. In those cases of extrahepatic and periampullary biliary tumors, in fact, usually highly undifferentiated, the histological characterization can be difficult, although a generic diagnosis of malignancy can be made. Potential complications due to forceps biopsy, such as disruption of the ductal wall with consequent bile leak and bleeding, cholangitis and pancreatitis, are reported [33]. Endoluminal forceps biopsy especially in patients with hemobilia or cholangitis, should therefore be performed in the remission phase of the disease, and, in any case, at least 5 days after biliary drainage, to avoid the complications related to manipulations into the biliary tree. No major complications were observed in this series, neither from percutaneous brushing nor from forceps biopsy. A transient hemobilia was observed in 5 patients who had undergone biopsy (10.6%). This series, including part of a previously analyzed smaller population [33] confirms that percutaneous endoluminal forceps biopsy has a very high sensitivity (92.1%), specificity (100%), accuracy (93.6%), PPV (100%), and NPV (75%) in the detection of malignant diseases (Table 2). The higher accuracy of biopsy over brushing is very clear, especially analyzing the data obtained in those patients in whom both techniques were performed. On the other hand, biopsy presents some disadvantages, such as the higher costs of the equipment and the difficult trackability across tight strictures or acute angles of the biliary ducts. A high diagnostic value was proven by the simple examination of tissue samplings coming from balloon dilatation of biliary or bilio-enteric anastomotic strictures (balloon surface sampling). Although the number of cases reported in this series is relatively small, it should be considered that it was possible to distinguish benign from malignant diseases in almost all the cases. Our suggestion, therefore is to associate a tissue collection from the balloon to bilioplasty, as a standard procedure when screening for malignant pancreatobiliary diseases is required. In this way. it is possible to save time and avoid risks related to further endobiliary procedures. Conclusion Patients with obstructive jaundice, who are candidates for biliary drainage, often come to the interventional radiologist still without a definitive diagnosis of the real nature of their disease, even after high-level cross-sectional imaging procedures, such as abdominal MR, MRCP or abdominal spiral-CT. When the clinical diagnosis needs to be histologically confirmed for further therapeutic choices, the transhepatic route can be successfully used for the intraductal sampling. Forceps biopsy is highly accurate under cholangioscopic guidance. As an alternative, repeated brushings can be performed under fluoroscopy. If a balloon blioplasty, for any reason, is performed, it is advisable to collect the tissue fragments on the balloon surface and send them for pathological evaluation. Competing interests None declared. Authors' contributions MR carried out the interventional procedures, participated in the design of the study and drafted the manuscript, FMS carried out the interventional procedures, VC defined the design of the study and performed the statistical analysis, LG collected the results of the procedures, LG participated in the histological analysis, AR participated in the design of the study and drafted the manuscript,, GG participated in the imaging evaluations, EP performed the patients follow-up, VD supervised the drafting of the study Figure 4 A 58 year old man, who 8 years ago underwent left hepatectomy and cholecistectomy, for complicated intrahepatic biliary stones, presented with jaundice and weight loss. Enhanced CT scan showed marked intrahepatic biliary dilation. Figure 5 Same patient as fig 4, at lower level, although, an extrinsic mass was not detected, the lumen of CBD appeared replacement by soft tissue mass density (arrows). Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments The study was supported by funds of La Sapienza University. ==== Refs Magnuson TH Bender JS Duncan MD Ahrendt SA Harmon JW Regan F Utility of magnetic resonance cholangiography in the evaluation of biliary obstruction J Am Coll Surg 1999 189 63 72 10401742 10.1016/S1072-7515(99)00082-4 Cozzi G Alasio L Civelli E Percutaneous intraductal sampling for cyto-histologic diagnosis of biliary duct strictures Tumori 1999 85 153 156 10426123 Tamada K Kurihara K Tomiyama T How many biopsies should be performed during percutaneous transhepatic cholangioscopy to diagnose biliary tract cancer? Gastrointest Endosc 1999 50 653 658 10536321 Farrell RJ Jain AK Brandwein SL The combination of stricture dilation, endoscopic needle aspiration, and biliary brushings significantly improves diagnostic yield from malignant bile duct strictures Gastrointest Endosc 2001 54 587 94 11677474 10.1067/mge.2001.118715 Cobb CJ Floyd WN Jr Usefulness of bile duct in the diagnostic management of patients with biliary tract obstruction Acta Citol 1995 29 93 100 Harell GS Anderson MF Berry PF Cytologic bile examination in the diagnosis of biliary neoplastic strictures Am J Roentgenol 1981 137 1123 1126 6274178 Kurzawinski T Deerv A Dooley J A prospective controlled study comparing brush and bile exfoliative cytology for diagnosing bile duct strictures Gut 1992 33 1675 1677 1487170 Saini S Imaging of the hepatobiliary tract N Engl J Med 1997 26 1889 1894 9197218 10.1056/NEJM199706263362607 Hann L Greatex K Bach A Cholangiocarcinoma at hepatic hilus: sonographic findings Am J Roentgenol 1997 168 985 989 9124155 Guibaud L Bret PM Reinhold C Atri M Barkun AN Bile duct obstruction and choledocholithiasis: diagnosis with MR cholangiography Radiology 1995 197 109 115 7568807 Chan YL Chan ACW Lam WWM Choledocholithiasis: comparison of MR cholangiography and endoscopic retrograde cholangiography Radiology 1996 200 85 89 8657949 Barish MA Yucel EK Soto JA MR cholangiography: efficacy of three-dimensional turbo spinecho technique Am J Roentgeno 1996 165 295 300 Regan F Fradin J Khazan R Bohlman M Magnuson T Choledocholithiasis: evaluation with MR cholangiography Am J Roentgenol 1996 167 1441 1445 8956574 Reinhold C Taourel P Bret PM Choledocholithiasis: evaluation of MR cholangiography for diagnosis Radiology 1998 209 435 442 9807570 Park MS Yu JS Kim YH Kim MJ Kim JH Lee S Acute cholecystitis: comparison of MR cholangiography and US Radiology 1998 209 781 785 9844674 Lee MG Lee HJ Kim MH Extrahepatic biliary diseases: 3D MR cholangiography compared with endoscopic retrograde cholangiopancreatography Radiology 1997 202 663 669 9051013 Regan F Smith D Khazan R MR cholangiography in biliary obstruction using half-Fourier acquisition J Comput Assist Tomogr 1996 20 627 632 8708069 10.1097/00004728-199607000-00024 Becker CD Grossholz M Becker M Mentha G de Peyer R Terrier F Choledocholithiasis and bile duct stenosis: diagnostic accuracy of MR cholangiopancreatography Radiology 1997 205 523 530 9356639 Adamek HE Albert J Weitz M A prospective evaluation of magnetic resonance cholangiography in patients with suspected bile duct obstruction Gut 1998 43 680 683 9824351 Varghese JC Farrell MA Courteney G Role of MR cholangiopancreatography in patients with failed or inadequate ECRP AJR Am J Roentgenol 1999 173 1527 33 10584796 Savader JS Prescott AC Lund BG Osterman AF Intraductal Biliary Biopsy: Comparison of three techniques JVIR 1996 7 743 750 8897345 Savader JS Lynch CF Radvany GM Kudrik TB Andrews TR Geschwind JF Single-specimen bile cytology: a prospective study of 80 patients with obstructive jaundice JVIR 1998 9 817 821 9756072 Harell GS Anderson MF Berry PF Cytologic bile examination in the diagnosis of biliary ducts neoplastic strictures Am J Roentgenol 1981 137 1123 1126 6274178 Rosch T Meining A Fruhmorgen S A prospective of the diagnostic accuracy of ERCP, MRCP, CT and EUS in biliary stricture Gastrointest Endosc 2002 55 870 876 12024143 10.1067/mge.2002.124206 Schöfl R Haefner M Wrba F Forceps biopsy and brush cytology during endoscopic retrograde cholangiopancreatography for the diagnosis of biliary stenoses Scand J Gastroenterol 1997 32 363 368 9140159 Rabinovitz M Zajko AB Hassaneim T Diagnostic value of brush cytology in the diagnosis of bile duct carcinoma: a study in 65 patients with bile duct strictures Hepatology 1990 12 747 752 2210678 Jaiwala J Fogel EL Sherman S Gottlieb K Flueckiger J Bucksot LG Leman GA Triple tissue sampling at ERCP in malignant biliary obstruction Gastrointest Endosc 2000 51 383 90 10744806 De Bellis M Fogel EL Sherman S Influence of stricture dilation and repeat brushing on the cancer detection rate of brush cytology in the evaluation of malignant biliary obstruction Gastrointest Endosc 2003 58 176 82 12872082 Stewart CJ Mills PR Carter R O'Donohue J Fullarton G Imrie CW Murray WR Brush Cytology in the assessment of pancreatico-biliary strictures: a review of 406 cases J Clin Pathol 2001 54 499 55 10.1136/jcp.54.6.449 Ryan ME Cytologic brushing of ductal lesions during ERCP Gastrointestinal Endosc 1991 37 139 142 Hall-Craggs MA Lees WR Fine needle aspiration biopsy: pancreatic and biliary tumors Am J Roentgenol 1986 147 399 403 3487961 Schenechter MS Doemeny JM Johnson OJ Biliary duct shave biopsy with use of the Simpson atherectomy catheter JVIR 1993 4 819 824 8281007 Rossi M Lemos A Bonaiuti P Amoruso M Petrone A Petrozza V Benvenuto A Rossi P Diagnosi strumentale degli itteri ostruttivi: spazzolamento versus biopsia Radiol Med 1997 93 230 235

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-581533933910.1186/1471-2407-4-58Research ArticleProtein p16 as a marker of dysplastic and neoplastic alterations in cervical epithelial cells Volgareva Galina [email protected] Larisa [email protected] Yulia [email protected] Georgy [email protected] Ella [email protected] Darya [email protected] Alexander [email protected] Dimitry [email protected] Valeriya [email protected] Fjodor [email protected] Institute of Carcinogenesis, N.N.Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow 115478, Russia2 P.A.Gertzen Institute of Oncology, Russian Ministry of Health, Moscow 125284, Russia3 Chair of Obstetrics and Gynecology, Ukrainian Medical Stomatological Academy, Poltava, Ukraine4 Kourion Therapeutics AG, Langenfeld, Germany5 Institute of Clinical Oncology, N.N.Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow 115478, Russia2004 31 8 2004 4 58 58 17 3 2004 31 8 2004 Copyright © 2004 Volgareva et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Cervical carcinomas are second most frequent type of women cancer. Success in diagnostics of this disease is due to the use of Pap-test (cytological smear analysis). However Pap-test gives significant portion of both false-positive and false-negative conclusions. Amendments of the diagnostic procedure are desirable. Aetiological role of papillomaviruses in cervical cancer is established while the role of cellular gene alterations in the course of tumor progression is less clear. Several research groups including us have recently named the protein p16INK4a as a possible diagnostic marker of cervical cancer. To evaluate whether the specificity of p16INK4a expression in dysplastic and neoplastic cervical epithelium is sufficient for such application we undertook a broader immunochistochemical registration of this protein with a highly p16INK4a-specific monoclonal antibody. Methods Paraffin-embedded samples of diagnostic biopsies and surgical materials were used. Control group included vaginal smears of healthy women and biopsy samples from patients with cervical ectopia. We examined 197 samples in total. Monoclonal antibody E6H4 (MTM Laboratories, Germany) was used. Results In control samples we did not find any p16INK4a-positive cells. Overexpression of p16INK4a was detected in samples of cervical dysplasia (CINs) and carcinomas. The portion of p16INK4a-positive samples increased in the row: CIN I – CIN II – CIN III – invasive carcinoma. For all stages the samples were found to be heterogeneous with respect to p16INK4a-expression. Every third of CINs III and one invasive squamous cell carcinoma (out of 21 analyzed) were negative. Conclusions Overexpression of the protein p16INK4a is typical for dysplastic and neoplastic epithelium of cervix uteri. However p16INK4a-negative CINs and carcinomas do exist. All stages of CINs and carcinomas analyzed are heterogeneous with respect to p16INK4a expression. So p16INK4a-negativity is not a sufficient reason to exclude a patient from the high risk group. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary test for early diagnostics of cervical cancer. ==== Body Background Cervical cancer makes up about 10–12% of total women cancers [1,2] with the level of mortality in Russian population 5.0 per 100000 [1]. The tendency is being observed for the past decades towards reduction of both incidence and mortality. It is mainly due to the population-wide screening protocols in developed countries which allow identifying early asymptomatic forms of cervical carcinomas. However some problems remain to be solved concerning early detection of this type of cancer. The main screening test for cervical cancer is the cytological smear staining technique developed by G. Papanicolaou [3] and known as Pap test. Despite evident success this test gives a substantial rate of both false-positive and false-negative results. Histological analysis of a biopsy sample, more laborious in preparation and study as compared with that of a cytological smear, is also not absolutely efficient owing to a substantial rate of interobserver discrepancies among expert pathologists examining the same material [4]. Infection with human papilloma viruses (HPV) belonging to so-called high-risk group is the main risk factor of cervical cancer incidence [2]. To detect high risk HPVs in epithelial cells of a patient polymerase chain reaction (PCR) has been applied during the past two decades [5]. However this highly sensitive technique cannot resolve the problem of early cervical carcinoma detection also so far as many early stage lesions regress and epithelial dysplasia (i.e. cervical intraepithelial neoplasms, CINs) and carcinomas appear only in a minor part of the persons in whose epithelium (on a smear) high risk HPVs had been detected [2]. For the recent years several research groups [4,6-16] including us [11] have dwelt on the protein p16INK4a for a possible supplementary marker of dysplastic and neoplastic cervical epithelium lesions. This protein belongs to the group of cyclin-dependent kinase Cdk4/6 inhibitors [17] and is encoded by tumor suppressor gene INK 4a (synonyms: MTS 1, CDKN2, INK4a/ARF). Gene INK4a plays an important role in the regulatory pathway Cdk-Rb-E2F. The product of this gene p16INK4a prevents pRb phosphorylation by inactivating Cdk4/6; pRb keeps on binding E2F transcription factors and as a result cells stay in G1 phase not passing to DNA replication. In various tumor types INK4a as a bona fide tumor suppressor undergoes homozygous deletions, is inactivated by point mutations, LOH or hypermethylation; p16INK4a expression is reduced or ceases under such conditions or the protein function may be impaired [18]. Peculiarity of cervical carcinomas is due to the ability of HPV oncoprotein E7 to interact with pRb and inactivate it [19]. As a result, the regulatory pathway Cdk-Rb-E2F is disrupted and the status of gene suppressor INK4a and its protein becomes of no importance for a cell so far as they function upstream of the site of breakage. Cells with thus inactivated pRb pass cell cycle checkpoint G1/S without any obstacle. Reciprocallity between status of pRb and that of p16INK4a commonly found in human permanent cell lines (including cervical cell line cultures) [17,20,21] as well as in primary tumor cells [9,22,23] served for a logical prerequisite of utilizing p16INK4a protein as a marker of premalignant and malignant cervical epithelium cells. Functionally active gene RB was shown to be able to negatively regulate the expression of INK4a on a transcriptional level, but details of this negative feed-back loop remain obscure [24]. To estimate the applicability of p16INK4a as a marker of dysplastic and neoplastic alterations in cervical epithelium cells we analyzed the expression of this protein utilizing a highly p16INK4a – specific monoclonal antibody. We examined 197 samples in total. The materials studied included: 1) samples of normal epithelium of healthy women (cytological vaginal smears), 2) diagnostic biopsy samples from patients with cervical ectopia, 3) samples of CINs of various stages, 4) samples of invasive cervical cancer, 5) samples from different normal utery body and cervix tissues from women with gynecological diseases not associated with dysplastic lesions of epithelium (diagnostic biopsies and surgical materials) and 6) samples of cells from 3 cervical carcinoma cell lines. We also compare our data with the results presented by other groups working in similar directions [4,6-10,12-16]. We demonstrate in the present work that overexpression of the protein p16INK4a is typical for some samples of dysplastic and neoplastic epithelium of cervix uteri. We have not found at least a single sample overexpressing p16INK4a among control samples. The portion of p16INK4a-positive samples increases in the following row: CIN I – CIN II – CIN III – invasive carcinoma. However p16INK4a-negative CINs and carcinomas have also been found. All stages of CINs and carcinomas analyzed turn out to be heterogeneous with respect to p16INK4a expression: side by side with the samples which express p16INK4a in 25% of cells or more we detect samples which are stained poorly or lack any staining. So p16INK4a-negativity does not seem to be a sufficient reason to exclude a patient from the high risk group when results of Pap-test, HPV detection by PCR or histological investigation warn about possibility of poor prognosis. As far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16INK4a may be regarded as a supplementary (optional) test for early diagnostics of cervical cancer. Methods Immunohisto-/immunocytochemical study was performed on 197 samples in total. Those included 6 samples from normal epithelium of healthy women (cytological vaginal smears taken during regular examination), as well as the following surgical and diagnostic biopsy materials: 37 samples from patients with cervical ectopia, 113 samples of CINs of different stages including cancer in situ, 26 samples of invasive cervical cancer (21 squamous cell carcinomas and 5 adenocarcinomas), 12 samples of normal tissues from uterus body (myometrium) and cervix from patients with different gynecological diseases, 3 cervical cell line samples taken for positive control (see below). Apart from 9 smears (which included 6 samples from healthy women and 3 samples taken from cell cultures which served as controls) the rest 188 materials were paraffin-embedded histological blocks. The quality of smears turned out to be satisfactory for immunochemical analysis (Fig. 1a and 1d). Neutral formalin-fixed paraffin embedded samples of biopsies and surgery materials were received from archives of N.N.Blokhin Cancer Research Center (Moscow), P.A. Gertzen Institute of Oncology (Moscow), Central Clinical Hospital (Moscow) and Fourth Clinical Hospital (Poltava, Ukraine). For immunohistochemical analysis 4–5 μm serial sections were transferred on Histobond slides with adhesive layer (SMT Geraetehandel GmbH, Germany). The first section was stained with hematoxylin-eosin for traditional morphological analysis and verification of diagnosis by not less than two independent pathomorphologists. Presence of high risk HPV in cervical CIN and carcinoma cells was verified by PCR [25]. Some of the squamous cell carcinomas were tested by Southern blot hybridization in addition to PCR. The results of both methods coincided completely. To detect high-risk HPV in adenocarcinomas Hybrid Capture 2 test (enabling to detect but not to discern HPV 16 and HPV 18) was used. The following portions of samples (out of those studied immunochemically) were analysed for high-risk HPV genetic material: CINs I – 9/51, CINs II – 9/32, CINs III – 19/24, invasive squamous cell carcinomas – 21/21, adenocarcinomas – 5/5. This study did not cover all CINs which we studied immunochemically due to the tiny size of most of those samples. Vaginal normal epithelium smears obtained during regular examination from healthy women were transferred on HistoBond slides. Absence of abnormal cells was confirmed in Cytology Department of Cancer Research Center on a parallel slide. Smears were fixed for 5 min in 10% formaldehyde, washed with flowing water, and processed for further development as described for deparaffinized histological slices [9]. Cells of cervical cell lines were cultured under standard conditions in DMEM supplemented with 10% of foetal bovine serum. For immunocytochemistry cells were taken during culture receeding, dropped in a culture medium on a Histobond slide, air-dried and then processed identically to smears from healthy women. Immunohistochemical staining was carried out using p16INK4a-specific monoclonal antibodies E6H4 (MTM Laboratories AG, Germany) according to protocol by Klaes et al [9]. Controls in the course of immunohistochemical studies were as follows Positive controls 1). In the beginning of the study as a whole cells of three cervical cell lines were stained first. They were SiHa (HPV16-positive), C33a and HT-3 (both HPV-negative); all of them had been characterized as expressing the protein p16INK4a in earlier immunocytochemical studies with the same antibody [9]. 2). One HT-3 cell slide was stained in parallels with the first series of biopsy materials. 3). Every next group of slides intended for staining compulsorily included the slide with one of the serial sections of the CINIII or invasive carcinoma sample which had been characterized as a p16INK4a-positive one in our previous studies (on preceeding cuts). The analysis of the given slide series was carried out only if the positivity of the control sample was confirmed. Thus neither slide series in the present study was completely p16INK4a-negative. Negative controls 1). One more serial cut made from p16INK4a-expressing material was included into every slide series meant for staining (as in variant 3 of positive controls, – see above) which was processed in a usual way but PBS was applied instead of p16INK4a-specific antibody. The results were regarded as valid if this slide was negative. 2). As an intrinsic negative control served adjacent to CIN or carcinoma normal tissues including stromal elements. In neither case did we find any staining in these tissues (fig. 1c). In addition to four categories of staining defined by Klaes et al. [9],- poor (less than 1% of stained cells,- figure 1b), sporadic (1–5% of stained cells, – figure 1b), focal (cell clusters were stained but not more than 25% of cells were positive, – figure 2a) and diffuse (more than 25% of cells were stained, – figures 1c and 2c,2f,2g,2h), we formed one more group (negative) of those samples which totally lacked any stained cells (Figures 1a, 2b,2d and 2e). We regarded those cells as stained in which p16INK4a was expressed in nuclei and/or in a cytoplasm. Results Various types of p16INK4a-specific staining of cervix uteri normal, dysplastic and cancer cells are presented in Fig. 1a,1b,1c,1d and Fig. 2a,2b,2c,2d,2e,2f,2g,2h. We did not register any staining in either of 6 smear samples taken from healthy women (Fig. 1a). An example of a very poor cytoplasmic staining in some separate CIN I cells with a more pronounced (sporadic, both nuclear and cytoplasmic) staining in the adjacent cancer in situ cells is shown in Fig. 1b. In one invasive carcinoma we detected cytoplasmic staining in the predominant majority of cancer cells while but sole nuclei turned out to be stained (Fig. 1c); the boundary between cancer and normal tissues coincided with the line at which the staining discontinued. As to the cervical cells cultivated in vitro taken for positive controls, in HT-3 cells the specific staining was strongly manifested both in nuclei and in a cytoplasm (Fig. 1d) while in SiHa and C33a cells it was exclusively cytoplasmic. It is not clear yet why in one and the same cervical cancer sample the protein p16INK4a (normally showing its activity in nuclei) may be detected but in a cytoplasm in the majority of cells while in a number of cells both in nuclei and cytoplasm, why in some cervical cell cultures it is found solely in cytoplasm and in other cervical cell lines – in nuclei also. We did not find at least a single sample with an exceptionally nuclear staining; similar were the results by other investigators [9]. With keeping in mind that subcellular location of p16INK4a-specific staining varies to such a degree we scored as positive every sample in which the staining was expressed either in a cytoplasm or in both nuclei and cytoplasm. The data of the analysis of the cervical epithelium preparations stained with monoclonal antibodies E6H4 are summarized in Table 1. In all 37 samples from patients with cervical ectopia p16INK4a-positive cells were observed with low frequencies: 34 samples were negative and 3 ones (8,1%) were stained poorly. In the group of CINs I 63% of the samples were p16INK4a-negative. In 7 samples (about 14%) sporadic or focal (Fig. 2a) type of staining was observed. Group of CINs II turned out to be highly heterogeneous in terms of the ratio of p16INK4a-positive cells as well. Most of these samples (68%, 26 out of 38, Fig. 2b) did not differ from the samples of normal epithelium, but the rest 12 samples we attributed to poor, sporadic or focal types. In neither sample of CIN I or CIN II did we observe diffuse staining for p16INK4a. Among the samples of CIN III about 54% (13 out of 24) were attributed to sporadic (Fig 1b), focal or diffuse (Fig. 2c) types. However every third CIN III sample was found to be p16INK4a-negative (Fig. 2d). As to invasive cancers, only 1 out of 26 samples (3.8%) lacked p16INK4a-positive cells (Fig. 2e). One sample expressed the marker poorly. In 24 cases sporadic, focal or diffuse staining was observed. Diffuse staining was registered in about 50% of these samples (Figs. 1c, 2f,2g,2h). Among 14 samples of invasive carcinomas which had been stored in paraffin blocks for 7 years 1 (7.1%) turned out to be p16INK4a-negative, 1 (7.1%) stained poorly, 3 (21.4%) stained sporadically, 4 (28.4%)-focally and 5 (35.8%) expressed diffuse staining. We have examined several cases with more than one type of lesion on the same slide. Examples are presented on Figures 1b and 2c. As a rule in combined cases p16INK4a expression was more pronounced in cells belonging to a more advanced lesion (Fig. 1b). All the HPV-tested samples of CINs and invasive squamous cell carcinomas were high-risk HPV-positive (data on CINs I and CINs II are shown in the additional file, results with CINs III and squamous cell carcinomas – in Table 2). High-risk HPVs were also found in all the samples of adenocarcinomas. In reference group composed of samples from normal uterus body and cervix uteri tissues obtained from patients with gynecological diseases (stromal and glandular tissues of the cervix from patients with cervical ectopia from which squamous cell epithelium had been fully cut off; myometrium of uterus body from the patient after surgery for cervical carcinoma) the results of staining with p16INK4a-specific antibodies were negative in all 12 cases (Table 3). Discussion Immunochemical detection of p16INK4a by monoclonal antibodies shows that the overwhelming majority of invasive cervical carcinoma samples differ from normal cervical epithelium of healthy women. We found no cases of sporadic, focal or diffuse staining with E6H4 antibodies among 6 vaginal smears from healthy women as well as among 37 samples from patients with cervical ectopia including those in which ectopically localized cervical epithelium koilocytes or condyloma had been registered. In addition, in 11 samples of normal glandular and stromal cervical tissues and one sample of uterus body normal myometrium tissue which were obtained from patients with gynecological disorders p16INK4a was not expressed at all. The predominant majority of invasive carcinoma samples were both high-risk HPV-positive and p16INK4a-expressing. However in 2 samples not expressing any p16INK4a or expressing it poorly high-risk HPV DNA sequences were detected by both PCR and Southern blot hybridization. These data are in a good agreement with those by Klaes et al., who described two samples of p16INK4a-negative but HPV-positive cervical cancer [9]. Among CIN III samples which were stained in our experiments poorly or lacked any staining (table 2, samples 1–6) high risk HPVs were found in 6 out of 6 tested. As to CIN I and CIN II samples expressing p16INK4a in less than 1% of cells (Additional file data, samples 1–5 and 10–14, respectively), we did not find any high-risk HPV-negative case in those groups either. Thus there seem to exist dysplastic and neoplastic lesions of cervix uteri which do not overexpress the protein p16INK4a but harbor high-risk HPV DNA. We confirm the data by Klaes et al. [9] that prolonged (for 7 years in our case) preservation of cervical carcinoma samples in paraffin blocks does not preclude the material from diffuse staining. Monoclonal antibodies E6H4 originally described by Klaes et al [9] had been tested by those authors among a number of clones of p16INK4a-specific monoclonal antibodies. The following commercially available clones had been taken: 1). DCS-50.1/H4-NA29 (Oncogene Research Products, Cambridge, MA), 2). 375P (Biogenex Laboratories, San Ramon, CA), 3). ZJ11 and JC8 (NeoMarkers, Freemont, CA), 4) 05–418 (Upstate Biotechnology, Lake Placid, NY) and 5). J175–405 (PharMingen, San Diego, CA). The results of the comparative experiments by Klaes at al [9] had demonstrated that clone E6H4 had turned out to be the most specific as inferred on the lowest level of unspecific staining on different tumor cell lines, both HPV-negative and positive. As in our experiments Klaes et al. [9] registered mainly cytoplasmic staining both in cells of permanent cell lines and in preneoplastic and carcinoma samples; an exclusive nuclear staining was not detected. We observed strong p16INK4a-positivity (sporadic or focal staining) in a comparatively small number of CIN I and CIN II samples (13–14 %). Among CINs III the portion of such samples increased up to 54,2%, with diffuse staining in every sixth case. The data we have obtained for CINs of all stages rather differ from the results by Klaes et al. [9]. All of CINs II and CINs III were stained as diffuse by Klaes et al. [9]. In the present study the group of CINs as a whole is much more heterogeneous. The reasons for those discrepancies are not quite clear yet. The population of Russian and Ukrainian patients whose materials were used in the present study was extremely heterogeneous with respect to age, nationality, etc. We cannot exclude that the discrepancies mentioned may be due to these factors as had been discussed earlier [26]. Nevertheless the coincidence between our results and the data by Klaes et al [9] concerning the general trend seems much more important: the increase of p16INK4a expression in dysplastic and neoplastic cervical epithelium in the course of progression. This trend is especially evident in our study when negative cases only are taken into consideration. All 6 samples were p16INK4a-negative in control group. Similar was the situation in the reference group: 12/12 negative. Among CINs I, CINs III and invasive carcinomas these indices made up 32/51 (63 %), 8/24 (33%) and 1/26 (4 %) respectively. The data presented herein allow us to conclude that hyperexpression of p16INK4a when detected immunohistochemically may be regarded as a marker of dysplastic and neoplastic lesions in cervical epithelium. Positive correlation between p16INK4a expression and morphological stage of the disease on the same slide (when combined cases with both dysplastic and neoplastic lesions were found) also favors this inference. In a number of recent communications immunochemical staining for p16INK4a was suggested to be performed on cytological smears [9,13-15]. Cytological approach has some advantages, so far as it enables a patient to avoid surgical intervention. That is why one may expect in the nearest future immunocytochemical version for detecting p16INK4a to become rather widely used not only as an addition to surgery but also for regular examinations in outpatient clinics. In this connection it seems important to estimate frequencies of a feasible false-negativity of the test. Keeping in mind that dysplastic lesions of all stages frequently regress and do not convert into invasive cervical carcinomas [2], we summarized the data by different groups on the frequencies of p16INK4a-negative invasive carcinoma samples (Table 4). We found that p16INK4a-negative cervical adenocarcinomas had been detected with frequencies which had varied from 0% up to 42,5% while p16INK4a-negative squamous cell carcinomas – with frequencies that had not exceeded 10,1%. Substantial variability of the data may be due to small numbers of samples analyzed (in some of the studies), to utilization of different types of monoclonal antibodies, to different criteria used by different research groups for the results interpretation, etc. It seems important to realize the proper place of immunocyto-/ immunohistochemical analysis of p16INK4a expression among previously developed tests for early detection of cervical carcinomas. In this connection the following points deserve mentioning. A pathologist in the course of common histological investigation usually registers some morphologic features such as zones of active mitotic divisions, multipolar mitoses, vicinity of condilomas and other formations. However when performing an immunocytochemical analysis on a smear a cytologist cannot bring in a correlation with morphological structure results of staining with p16INK4a-specific antibodies in separate cells. According to the results of the present study about 33% of CINs III as well as about 5% of invasive squamous cell carcinomas of cervix uteri do not differ from normal cervical epithelium with respect to p16INK4a expression. Thus immunochemical staining for p16INK4a (in both cyto- and histochemical versions) does not seem to be the approach that can help to fully overcome absolutely all existing ambiguities of cervical cancer early diagnostics. As to possible sources of false positivity of immunochemical detection of the protein p16INK4a the study by Agoff et al [16] deserve mentioning. According to this communication in 10 out of 10 endometrial biopsy samples studied endometrial cells were positively stained with the p16INK4a – specific antibody E6H4. So far as endometrial cells may occur on vaginal smears the test does not seem to enable one to fully avoid false positivity. Conclusions Overexpression of the protein p16INK4a encoded by tumor suppressor gene INK4a is a characteristic of displastic and neoplastic alterations of cervical epithelium. The portion of p16INK4a-positive samples increases in the following row: CIN I – CIN II – CIN III – invasive carcinoma. All stages of CINs and carcinomas analysed are heterogeneous with respect to p16INK4a expression: side by side with the samples which expressed p16INK4a in 25% of cells or more we detected samples which were stained poorly or lacked any staining. According to the data we present herein p16INK4a-negative cervical neoplasms and carcinomas do exist. Thus the lack of p16INK4a-positive cells in samples from different types of cervical lesions should not be regarded as a sufficient reason for excluding a patient from the high-risk group. Despite this as far as normal cervical epithelium is p16INK4a-negative and the ratio p16INK4a-positive/ p16INK4a-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16INK4a may be regarded as an additional (optional) test for early detection of precancerous lesions in cervical epithelium. Competing interests none declared Author's contributions F.K., D.S., G.V. and G.F. contributed to study conception and design; E.K. and A.B. delt with sample obtaining and preliminary diagnosis; Yu.A., G.F. and V.E. were independent pathologists who confirmed the diagnosis; G.V., L.Z. and D.G. performed immunohistochemical staining and analysis; A.B. carried out HPV typing; G.V. drafted the manuscript. All authors read and approved the final version. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Data on high risk HPV genome detection by PCR in CIN I and CIN II samples. the data are presented on high-risk HPV genetic material detection in 9 CIN I and 9 CIN II samples. Click here for file Acknowledgements The authors thank Drs N.P.Kisseljova and N.N.Mazurenko for promoting discussion of the results. We appreciate assistance of Drs. A.Petrov and A.Petrenko in preparing the manuscript. The work was supported in part by FIRCA grant NIH 0600-070-c223 as well as by the Russian Foundation of Basic Science (grant No 02-04–48271) and Russian Ministry of Science (grant N 1877). Figures and Tables Figure 1 Different types of cervical cell staining with the p16INK4a-specific antibodies. a. Normal epithelium (smear): negative staining. b. CIN I (indicated with a dotted arrow) and cancer in situ (solid arrows): a very poor cytoplasmic staining in separate CIN I cells and sporadic staining in cancer in situ. c. Invasive squamous cell carcinoma. Diffuse cytoplasmic staining with the sole cell expressing p16INK4a in the nucleus (solid arrow). The boundary with adjoining normal tissue is marked with a dotted arrow. d. HT3 cells (smear) with both nuclear and cytoplasmic subcellular location of the positive staining. Figure 2 Cervical tissue samples after immunohistochemical staining with p16INK4a-specific antibodies. a. CIN I. Focal staining. b. CIN II. Negative staining. c. Cancer in situ (indicated with an arrow) and CIN III. Diffuse staining. d. CIN III. Negative staining. e. Squamous cell carcinoma. Negative staining. f. Squamous cell carcinoma. Diffuse staining. g. Squamous cell carcinoma embol. Diffuse staining. h. Adenocarcinoma. Diffuse staining. Table 1 p16INK4a EXPRESSION IN NORMAL, DYSPLASTIC AND NEOPLASTIC EPITHELIAL CELLS OF CERVIX UTERI STAINING* MATERIALS number of samples negative poor sporadic focal diffuse Normal epithelium (smears) 6 6 (100) - - - - Cervical ectopia including 37 34 (91,9) 3 (8,1) - - - C.e. + coilocytosis 10 9 1 - - - C.e. + condyloma 2 1 1 - - - Dysplasia: CINs I 51 32 (62,7) 12 (23,5) 6 (11,8) 1 (2,0) - CINs II 38 26 (68,4) 7 (18,4) 4 (10,5) 1 (2,7) - CINs III including cancer in situ 24 8 (33,3) 3 (12,5) 7 (29,2) 2 (8,3) 4 (16,7) Invasive carcinomas including squamous cell carcinomas adenocarcinomas 26 1 (3,8) 1 (3,8) 4 (15,5) 7 (26,9) 13 (50,0) Squamous cell carcinomas 21 1 (4,8) 1 (4,8) 3 (14,3) 6 (28,5) 10 (47,6) Adenocarcinomas 5 - - 1 (20,0) 1 (20,0) 3 (60,0) * percent is given in parenthesis Table 2 Data on high risk HPV genome detection by PCR in CIN III and invasive squamous cell carcinoma samples Sample No Type of immunochemical staining HPV type CINs III 1 negative 16 2 negative 18 3 negative 16 4 poor 16 5 poor 16 6 poor 16 7 sporadic 16 8 sporadic 16 9 sporadic 16 10 sporadic 16 11 sporadic 16 12 sporadic 16 13 sporadic 16 14 focal 16 15 focal 16 16 diffuse 16 17 diffuse 16 18 diffuse 18 19 diffuse 18 invasive squamous cell carcinomas 20 negative 16* 21 poor 16* 22 sporadic 16* 23 sporadic 16 24 sporadic 16* 25 focal 16* 26 focal 16 27 focal 16 28 focal 18 29 focal 16 30 focal 16 31 diffuse 16 32 diffuse 16 33 diffuse 16 34 diffuse 66 35 diffuse 16 36 diffuse 16 37 diffuse 16 38 diffuse 16 39 diffuse 18 40 diffuse 16 + 18 * confirmed by Southern blotting Table 3 STAINING OF THE REFERENCE MATERIALS WITH p16INK4a-SPECIFIC MONOCLONAL ANTIBODIES MATERIALS number of samples negative poor sporadic focal diffuse Normal tissues, including 12 12 - - - - Uterus body (myometrium) 1 1 - - - - Stromal and glandular tissues of cervix uteri 11 11 - - - - Table 4 THE SHARE OF p16INK4a-NEGATIVE SAMPLES AMONG CERVICAL CARCINOMAS (LITERATURE DATA) Research group (reference) The share (per cent) of p16INK4a-negative samples among adenocarcinomas: Lu et al, 1998 (7) 17/40 (42.5%) Sano et al, 1998 (8) 4/15 (26.7%) Milde-Langosch et al, 2001(10) 8/58 (13,8%) Klaes et al, 2001(9) 1/7 (14.3%) Saqi et al, 2002 (13) 0/2 (0%) Murphy et al, 2003 (14) 0/2 (0%) Negri et al, 2003 (15) 0/18 (0%) Agoff et al, 2003 (16) 2/7 (28.6 %) Present study 0/5 (0%) among squamous cell carcinomas: Wong et al, 1997 (6) 8/79 (10.1%) Sano et al, 1998 (8) 1/39 (2.8%) Klaes et al, 2001 (9) 1/53 (1.9%) Sano et al, 2002 (12) 0/34 (0%) Klaes et al, 2002 (4) 0/46 (0%) Saqi et al, 2002 (13) 0/1 (0 %) Murphy et al, 2003 (14) 0/8 (0%) Agoff et al, 2003 (16) 4/46 (8.7%) Present study 1/21 (4.8%) ==== Refs Zaridze DG Zaridze DG Epidemiology and etiology of malignant tumors In Carcinogenesis 2000 Moscow: Nauchny Mir 21 56 Russian Zur Hausen H Papillomaviruses and cancer: from basic studies to clinical applications Nature Reviews Cancer 2002 2 342 350 12044010 10.1038/nrc798 Papanicolaou GN Science 1942 95 438 439 Klaes R Benner A Friedrich T Ridder R Herrington S Jenkins D Kurman RJ Schmidt D Stoler M von Knebel Doeberitz M P16INK4a immunohistochemistry improves interobserver diagnosis of cervical intraepithelial neoplasia Am J Surg Pathol 2002 26 1389 1399 12409715 10.1097/00000478-200211000-00001 Walboomers JMM Melkert PWJ Van Den Brule AJC Snijders PJF Meijer CJLM Herrington CS, McGee J Application of polymerase chain reaction for screening of Human Papillomaviruses in cytopathologies of the cervix uteri Molecular Diagnostic Pathology. A practical approach 1999 Moscow: Mir 474 495 Wong YF Cheung TK Chung TKH Chan MKM Chang AMZ Expression of p16INK4a and Rb genes in cervical neoplasm J Lower Genital Tract Dis 1997 1 240 244 Lu X Toki T Konishi I Nikaido T Fujii S Expression of p21WAF1/CIP1 in adenocarcinoma of the uterine cervix: a possible immunohistochemical marker of a favourable prognosis Cancer 1998 82 2409 2417 9635534 10.1002/(SICI)1097-0142(19980615)82:12<2409::AID-CNCR15>3.3.CO;2-R Sano T Oyama T Kashiwabara K Fukuda T Nakajima T Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions Am J Pathol 1998 153 1741 1748 9846965 Klaes R Friedrich T Spitkovsky D Ridder R Rudy W Petry U Dallenbach-Hellweg G Schmidt D von Knebel Doeberitz M Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri Int J Cancer 2001 92 276 284 11291057 10.1002/ijc.1174 Milde-Langosch K Riethdorf S Kraus-Poppinghaus A Riethdorf L Loning T Expression of cyclin-dependent kinase inhibitors p16MTS1, p21WAF1, and p27KIP1 in HPV-positive and HPV-negative cervical adenocarcinomas Virchows Arch 2001 439 55 61 11499840 10.1007/s004280100439 Volgareva GM Zavalishina LE Frank GA Andreeva YuYu Petrov AN Kisseljov FL Spitkovsky DD Expression of protein marker p16INK4a in uterine cervical cancer Arkh Patol 2002 64 22 24 Russian 11889694 Sano T Masuda N Oyama T Nakajima T Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia Pathol Int 2002 52 375 83 12100520 10.1046/j.1440-1827.2002.01359.x Saqi A Pasha TL McGrath CM Yu GH Zhang P Gupta P Overexpression of p16INK4a in liquid-based specimens (SurePath trade mark) as marker of cervical dysplasia and neolpasia Diagn Cytopathol 2002 27 365 370 12451568 10.1002/dc.10205 Murphy N Ring M Killalea AG Uhlmann V O'Donovan M Mulcahy F Turner M McGuinness E Griffin M Martin C Sheils O O'Leary JJ P16INK4a as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrep smears J Clin Pathol 2003 56 56 63 12499437 10.1136/jcp.56.1.56 Negri G Egarter-Vigl E Kasal A Romano F Haitel A Mian C P16INK4a is a useful marker for the diagnosis of adenocarcinoma of the cervix uteri and its precursors Am J Surg Pathol 2003 27 187 193 12548164 10.1097/00000478-200302000-00006 Agoff SN Lin P Morihara J Mao C Kiviat NB Koutsky LA P16INK4a expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types Mod Pathol 2003 16 665 673 12861062 10.1097/01.MP.0000077518.78046.0C Serrano M Hannon GJ Beach D A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4 Nature 1993 366 704 707 8259215 10.1038/366704a0 Ruas M Peters G The p16INK4a/CDKN2A tumor suppressor and its relatives [review] Biochim Biophys Acta 1998 1378 F115 F177 9823374 10.1016/S0304-419X(98)00017-1 Dyson N Howley PM Munger K Harlow E The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product Science 1989 243 934 937 2537532 Tam SW Shay JW Pagano M Differential expression and cell cycle regulation of the cyclin-dependent kinase 4 inhibitor p16INK4a Cancer Res 1994 54 5816 5820 7954407 Khleif SN DeGregori J Jee CL Otterson GA Kaye FJ Nevins JR Howley PM Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity Proc Natl Acad Sci USA 1996 93 4350 4354 8633069 10.1073/pnas.93.9.4350 Geradts J Kratzke R Niehans GA Lincoln CE Immunohistochemical detection of the cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) product p16INK4a in archival human solid tumors: correlation with retinoblastoma protein expression Cancer Res 1995 55 6006 6011 8521382 Kratzke RA Greatens TM Rubins JB Maddaus MA Niewoehner DE Niehans GA Geradts J Rb and p16INK4a expression in resected non-small cell lung tumors Cancer Res 1996 56 3415 3420 8758904 Lowe SW Sherr CJ Tumor suppression by Ink4a-Arf: progress and puzzles Curr Opin Genet Dev 2003 13 77 83 12573439 10.1016/S0959-437X(02)00013-8 Samoylova EV Shaikhaiev GO Petrov SV Kisseljova NP Kisseljov FL HPV infection in cervical-cancer cases in Russia Int J Cancer 1995 61 1 5 7705920 Kisseljov FL Kisseljova NP Kobzeva VK Gritsko TM Semenova LA Pavlova LS Klaes R von Knebel Doeberitz M State of Human Papilloma virus DNA in Cervical Carcinomas Molecular Biology 2001 35 399 404 10.1023/A:1010430913634

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==== Front BMC CancerBMC Cancer1471-2407BioMed Central London 1471-2407-4-591534166810.1186/1471-2407-4-59Research ArticleHER2 expression in cervical cancer as a potential therapeutic target Chavez-Blanco Alma [email protected] Victor [email protected] Aurora [email protected] Teresa [email protected] Myrna [email protected] Lucely [email protected] Silvia [email protected]ñas-Gonzalez Alfonso [email protected] Unidad de Investigación Biomédica en Cáncer. Instituto Nacional de Cancerología-Instituto de Investigaciones Biomédicas, UNAM, Av. San Fernando No. 22, Tlalpan 14080, Mexico City2 Department of Pathology, Instituto Nacional de Cancerología; Av. San Fernando No. 22, Tlalpan 14080, Mexico City3 Division of Clinical Research, Instituto Nacional de Cancerología. Av. San Fernando No. 22, Tlalpan 14080, Mexico City2004 1 9 2004 4 59 59 14 4 2004 1 9 2004 Copyright © 2004 Chavez-Blanco et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Trastuzumab, a humanized monoclonal antibody against the HER2 receptor is currently being used in breast and other tumor types. Early studies have shown that a variable proportion of cervical carcinoma tumors overexpress the HER2 receptor as evaluated by diverse techniques and antibodies. Currently it is known that a tumor response to trastuzumab strongly correlates with the level of HER2 expression evaluated by the Hercep Test, thus, it seems desirable to evaluate the status of expression of this receptor using the FDA-approved Hercep Test and grading system to gain insight in the feasibility of using trastuzumab in cervical cancer patients. Methods We analyzed a series of cervical cancer cell lines, the primary tumors of 35 cases of cervical cancer patients and four recurrent cases, with the Hercep Test in order to establish whether this tumor type overexpress HER2 at level of 2+/3+ as trastuzumab is currently approved for breast cancer having such level of expression. Results The results indicate that only 1 out of 35 primary tumors cases overexpress the receptor at this level, however, two out of four recurrent tumors that tested negative at diagnosis shifted to Hercep Test 2+ and 3+ respectively. Conclusions The low frequency of expression in primary cases suggests that trastuzumab could have a limited value for the primary management of cervical cancer patients, however, the finding of "conversion" to Hercep Test 2+ and 3+ of recurrent tumors indicates the need to further evaluate the expression of HER2 in the metastatic and recurrent cases. ==== Body Background Cervical carcinoma is a leading cause of death in women of reproductive age worldwide, particularly in developing countries. While curable in early stages, the treatment results of locally advanced disease are unsatisfactory. The current standard of treatment -cisplatin-based chemoradiation- fails to cure at least 15% to 45% of bulky IB to IIIB patients, and in addition, multimodality treatment incorporating chemotherapy, surgery and radiation at its best is unlikely to substantially increase the cure rate. Because of this, the logical step to follow is the testing of molecular targeted therapies trying to improve the prognosis of cervical cancer patients [1]. Human papillomavirus infection is recognized as the stronger etiological factor for the development of this tumor; however, overexpression of the epidermal growth factor receptor family members is also common and seems to play an important oncogenic role [2]. HER2 (also known as c-erbB-2) is a transmembrane receptor protein with tyrosine kinase activity that belongs to this family and it is overexpressed in a number of solid tumors. Its overexpression and prognostic significance in breast cancer led to the development and approval of the use of trastuzumab (Trastuzumab, Genentech, South San Francisco, CA), a recombinant monoclonal antibody to HER2, for the treatment of patients with metastatic breast carcinomas overexpressing HER2 [3]. Until more recently, poor standardization in HER2 status evaluation precluded reliable comparison of overexpression rates in different tumors. A source of variability in results not only comes from methodological variations in tissue processing (time to fixation, duration of fixation, denaturation, heating, antigen retrieval, the staining procedure) and grading scores but also from the antibody used. This issue was addressed by Press et al., who showed extremely variable results in 187 breast cancer specimens evaluated with 7 polyclonal and 21 monoclonal antibodies [4]. However, standardized methodologies have been introduced recently for these analyses, and have identified frequencies of 51%, 44%, 26% and 25% in Wilm's tumor, bladder, pancreatic and breast carcinoma, respectively. Other tumors tested had frequencies below 20% [5]. Before the introduction of the Hercep Test, it was known that a variable subset of cervical carcinomas ranging from 8% to 77% express HER2 as evaluated by diverse methods [6-14] and that in some studies its overexpression has shown to confer a worse prognosis [7-9,13]. Because these results on HER2 expression in cervical cancer were obtained before the standardization required in breast cancer, we wanted to investigate the expression status of HER2 using the Hercep Test in a series of cervical carcinoma cell lines, primary tumors of locally advanced cervical cancer cases and in four recurrent tumors of these patients. Methods Tumor specimens Thirty-five paraffin-embedded tumor tissues from patients FIGO staged as IB2 to IIIB, treated with standard radiation concurrent with weekly cisplatin. Diagnosis was made on the basis of routine hematoxilin-eosin examination under light microscopy according to the World Health Organization criteria. Tumor specimens at diagnosis were taken before any treatment was instituted whereas the tumors samples from the four recurrent cases were also taken before patients received any second line therapy. Cell lines and reagents DMEM culture media and Fetal Calf Serum were purchased from Gibco BRL Life Technologies (Grand Island, New York). HeLa, CasKi, SiHa and C33A carcinoma cell lines were obtained from the ATCC. The cell line ViBo established from a Mexican patient with cervical cancer was kindly provided by Dr. Monroy (FES Zaragoza, UNAM, Mexico City). Cells were grown in DMEM supplemented with 10% FCS at 37°C and 5% CO2. Cell lines were grown on two-chamber polysterene vessel Falcon® (Becton Dickinson, NJ.) and subsequently formalin-fixed for 24 hrs at room temperature, then rehydrated in graded ethanol. Afterwards immunochemistry was performed as below described. Hercep test Hercep Test was performed following the manufacturer's guidelines of HER2 protein expression as follows. Sections were deparaffinized in xylene and rehydrated through graded ethanols to distilled water. The sections were immersed in Dako Epitope Retrieval Solution (10 mM citrate buffer, pH6) that had been preheated to 95°C in a water bath and then heat-treated at 95°C for 40 min. After a 20-minute cooldown period at room temperature, the sections were washed with Dako Wash Buffer, a procedure that followed every subsequent incubation. Endogenous peroxidase was blocked with Dako Blocking Buffer (0.3% hydrogen peroxide containing 15 mM sodium azide) for 5 min at room temperature. The sections were incubated with the primary polyclonal antibody, an affinity-purified rabbit antihuman HER2 antibody supplied in the kit, for 30 min at room temperature. Bound primary antibody was labeled by incubating the slides with the Dako Visualization reagent (horseradish peroxidase-labeled dextran polymer conjugated to affinity-purified goat antirabbit immunoglobulins in Tris-HCl) for 30 min. Color development was achieved with 3,3'-diaminobenzidine (DAB) for 10 min. The sections were counterstained with hematoxylin and eosin. To confirm validation of the staining run, control cell slides, which were provided in the kit and consisted of three pelleted, formalin-fixed, paraffin-embedded human breast cell lines with known HER2 positivity (MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were also stained simultaneously. In the negative controls, the primary antibody was replaced by normal rabbit serum (Dako Negative Control Reagent) for the HER2 primary antibody. The antibody used in Hercep Test did not exhibit cross-reactivity to HER3 and HER4 in western blot analysis. Following the FDA scoring guidelines for breast carcinomas, only membrane staining intensity and pattern were evaluated using the 0–3+ scale as illustrated in the Hercep Test kit scoring guidelines (0 for no staining at all or membrane staining in less than 10% of the tumor cells; 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells; 2+ for moderate staining of the complete cell membrane in more than 10% of the tumor cells; 3+ for intense staining of the complete membrane in more than 10% of the tumor cells). The analysis was performed by a pathologist (VP-S) familiarized in the use of Hercep Test for breast cancer patients. In accordance with the Hercep Test kit guide, HER2 overexpression was assessed as negative for scores of 0 or 1+ and positive for scores of 2+ and 3+. FISH in the four recurrent cases Amplification of Her-2/neu was evaluated using the Path-Vysion DNA Probe Kit (Vysis), which uses a dual-color probe for determining the number of copies of both Her-2/neu (orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturer's instructions with a few minor modifications. Slides containing 5μ thick paraffin-embedded tissue sections of studied cervical tumor cases and a known Her-2/neu amplified breast tumor were placed on a slide warmer overnight at 58°C, followed by deparaffinization in Xilol, dehydration in 100 ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0.2 N hydrochloric acid for 20 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 10 minutes, washed in Vysis wash buffer, and dried on the slide warmer. The slides were then immersed in 10% buffered formalin at room temperature for 10 minutes, immersed in Vysis wash buffer, and dried on the slide warmer. Sections were denatured by placing the slides in formamide for 5 minutes at 72°C followed by dehydration in 70, 85 and then 100% ethanol. Slides were then dried on a slide warmer, and 10 μl of probe was applied. They were then coverslipped, sealed and placed in a prewarmed humid incubation chamber at 37°C for 21 hours. This was followed by immersion in prewarmed postwash solution at 72°C for 2 minutes. The slides were air-dried, and a 4',6-diamidino-2-phenylindole (DAPI) counterstain was applied. The scoring system used is described in detail in the manufacturer's instruction. A minimum of 60 nuclei were scored by each of 2 observers using a Zeiss Axioskop-2 fluorescent microscope with V.2 filter. The ratio of Her-2/neu signals (orange) to chromosome 17 centromere signals (green) was determined with ratios of <2.0 considered nonamplified and those ≥ 2.0 amplified. Results The immunohistochemical expression of HER2 in the primary tumors of 35 patients at diagnosis was evaluated. Accordingly, these patients had no received any previous anticancer therapy; their mean age was 40.8 years; 5 were staged as IB2-IIA, 14 as IIB and 16 as IIIB; 31 and 4 were histologically classified as squamous and adeno/adenosquamous respectively. Overexpression of HER2 was demonstrated in only one out of 35 cases, which had a score of 3+ (not shown). The remaining cases were interpreted as negative [score of 0]. The case with HER2 overexpression at diagnosis was a 56-year old woman diagnosed with a FIGO stage IIB large cell poorly differentiated squamous cell carcinoma, who received treatment with 6 weekly courses of cisplatin concurrent with external beam radiation and brachytherapy. She is currently free of disease at 44 months of follow-up. At a median follow-up time 40 months, seven patients have relapsed. HER2 expression at recurrence could only be analyzed in four of these seven relapsed patients in whom whose recurrent disease was histopathologically confirmed. Two of these four tested positive with a staining intensity of 2+ and 3+ respectively, (Figures 1 and 2), both cases were squamous cell carcinomas and tested negative in the pretreatment surgical specimen The five cervical cancer cell lines were negative. None of the four recurrent cases tested by FISH were HER2 amplified (Figure 3). Discussion Molecular targeted therapies are currently being tested in a variety of tumor types with promising results. Because HER2 overexpression occurs and is related to a worse prognosis in cervical cancer, [7-9,13], its blockade with trastuzumab could potentially have therapeutic value. This monoclonal antibody is currently widely used in metastatic breast cancer and is being evaluated in an adjuvant setting as well as in a variety of tumor types [3,15-17], Based on the fact that the efficacy of this antibody is strongly associated to the level of HER2 expression in the primary tumor, the FDA approved the Hercep Test in the aim to grade the expression level so that only those patients whose tumors exhibit a 2+/3+ levels are candidates to trastuzumab therapy, though currently in most centers, tumors expression of 2+ is considered undeterminate therefore these cases are also evaluated by FISH analysis [18]. Previous reports on cervical cancer using non-standardized methods for HER2 expression showed that up to 77% of cases express the receptor and that in general HER2 expression predominates in adenocarcinoma and adenosquamous carcinoma histologies [6-14] In this work, using the Hercep Test with its corresponding guidelines for evaluation, we found contrastating results as none of the cell lines expressed HER2 and only a single tumor of squamous histology (1 out of 35) expresses this oncoprotein at a level of 3+. Such a discrepancy does not seem to be limited to this tumor type. For instances, in ovarian clear cell carcinoma a 43% of overexpression was reported utilizing systems other than Hercep Test [19], however, when evaluated with this standardized test, only 1 out of 17 tumors expressed 3+ [20]. Likewise, the proportion of patients with 2+/3+ expression level of HER2 with the Hercep Test is uniformly low in tumor such as lung [21], colorectal carcinomas [22], gallbladder [23], and melanoma [24]. A variety of factors such as the kind of antibody used, the technique per se, and scoring criteria may explain such phenomenon and its clarification requires further studies. A recent paper by Bellone et al., have reported that a substantially higher proportion of cervical cancer cells lines either established from fresh tumors or commercial ones (including CasKi, SiHa, HeLa and C33A which are negative by immunochemistry) express the receptor when evaluated by flow-cytometry and are growth inhibited when incubated with trastuzumab or trastuzumab plus IL-2 [25]. These data led them to suggest the targeting the HER in cervical cancer could be more effective than the indicated by low immunohistochemical expression [25]. However, it is largely known that in breast [26] and more recently in lung cancer [27], tumor responses are almost confined to those with a 3+ level of expression. For instances, in a recent published study in 111 assessable breast cancer patients, the response rate to single agent trastuzumab for those expressing 3+ versus 2+ was 35% and 0% respectively [26], while in lung cancer, a phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive patients, yet there was not overall differences in response between both arms, the benefit was limited to those with 3+ of expression with the Hercep Test. Accordingly, five out of six patients with such level of expression receiving trastuzumab plus gemcitabine-cisplatin showed response [27]. These data argue against the potential usefulness of trastuzumab in cervical cancer patients with HER expression that can only be detected by flow cytometry [25]. On the other hand, the HER2 expression in breast cancer is relatively stable, with 95% concordance between the HER2 status of primary and metastatic lesions, being rare a shift from positivity in the primary to negativity in the metastases [5]. Conversely, 6% of breast cancer patients whose primary tumors are HER2 negative, convert to high expression (Hercep Test 3+) in their metastases [28]. Such behavior is in line with experimental observations that receptor activation potentiates tumor cell motility, protease secretion and invasion, and also modulates cell cycle checkpoint function, DNA repair, apoptotic responses and multidrug resistance [29,30]. The findings of "conversion to positive" in cells of recurrent cervical tumors showed by us and other authors [25,31] strongly suggest that expression of HER2 may have a role in tumor resistance and progression as shown in experimental models, and therefore its targeting in recurrent cervical cancer could have therapeutic value. It is remarkable the finding that none of the four recurrent cases, including the two that converted to IHC positive analyzed by FISH showed HER2 gene amplification. This result is unlikely to be a false negative as the green signal was perfectly observed in most of the cells. Previous studies in cervical cancer have shown that the frequency of gene amplification as determined by FISH is low irrespective of tumor histology. Mark et al., reported only 2 out of 23 cases amplified using the Her-2/neu FISH probe (Vysis, Inc., Downers Grove, IL) both of which were adenocarcinomas [32]. In a more recent study looking at DNA copy number of cervical adenocarcinomas, it was found that despite more than 50% of patients had chromosome 17q copy number gains, only 9% (2 out of 22) of these tumors showed an HER-2/neu protein over-expression at the level of 2+ with the Hercep test. These findings suggest that amplification of HER-2/neu is rare in cervical adenocarcinomas and that low level chromosome 17q copy number gains are not associated with HER-2/neu overexpression [33]. Such overexpression without gene amplification could result from transcriptional deregulation leading to increased receptor expression [34] and is not a rare phenomenon in breast carcinoma [35]. Conclusions In conclusion, our study suggest that the clinical usefulness of anti-HER2 antibodies in the primary treatment of cervical cancer patients may be limited due to the low frequency of HER overexpression, nevertheless, it is desirable to further test a larger number of recurrent cervical cancer patients by IHC and FISH analyses regardless of the histological type, as a start point for clinical trials design using trastuzumab. Competing interests None declared. Authors'contributions A C-B, AG-F, carried out the tissue culture work and immunohistochemical analysis; VP-S and T V-C interpreted the histological data; MC critically analyzed and participated in manuscript; CP, contribute with the clinical data; SV performed the FISH analysis; and AD-G conceived the study and wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We thank to Virginia Enriquez for her technical support. Figures and Tables Figure 1 Photomicrography of a recurrent tumor positive at a intensity of 2+. (200X). Figure 2 Photomicrography of a recurrent tumor positive at a intensity of 3+. (200X). Figure 3 Photomicrography of the FISH analysis of a negative case of a recurrent tumor that was Hercep Test-positive, and a control positive case of breast carcinoma. ==== Refs Dueñas-Gonzalez A Cetina L Mariscal I de la Garza J Modern management of locally advanced cervical carcinoma Cancer Treat Rev 2003 29 389 399 12972357 10.1016/S0305-7372(03)00068-9 Chang JL Tsao YP Liu DW Han CP Lee WH Chen SL The expression of type I growth factor receptors in the squamous neoplastic changes of uterine cervix Gynecol Oncol 1999 73 62 71 10094882 10.1006/gyno.1998.5301 Horton J Trastuzumab use in breast cancer: clinical issues Cancer Control 2002 9 499 507 12514568 Press MF Hung G Godolphin W Slamon DJ Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression Cancer Res 1994 54 2771 2777 7909495 Menard S Casalini P Campiglio M Pupa S Agresti R Tagliabue E HER2 overexpression in various tumor types, focussing on its relationship to the development of invasive breast cancer Ann Oncol 2001 12 S15 19 11521715 10.1023/A:1011151505425 Kristensen GB Holm R Abeler VM Trope CG Evaluation of the prognostic significance of cathepsin D, epidermal growth factor receptor, and c-erbB-2 in early cervical squamous cell carcinoma. An immunohistochemical study Cancer 1996 78 433 440 8697388 10.1002/(SICI)1097-0142(19960801)78:3<433::AID-CNCR9>3.3.CO;2-E Oka K Nakano T Arai T c-erbB-2 Oncoprotein expression is associated with poor prognosis in squamous cell carcinoma of the cervix Cancer 1994 73 664 671 7905364 Kihana T Tsuda H Teshima S Nomoto K Tsugane S Sonoda T Matsuura S Hirohashi S Prognostic significance of the overexpression of c-erbB-2 protein in adenocarcinoma of the uterine cervix Cancer 1994 73 148 153 7506116 Niibe Y Nakano T Ohno T Suzuki Y Oka K Tsujii H Prognostic significance of c-erbB-2/HER2 expression in advanced uterine cervical carcinoma with para-aortic lymph node metastasis treated with radiation therapy Int J Gynecol Cancer 2003 13 849 855 14675323 10.1111/j.1525-1438.2003.13397.x Lee JS Kim HS Jung JJ Lee MC Park CS Expression of vascular endothelial growth factor in adenocarcinomas of the uterine cervix and its relation to angiogenesis and p53 and c-erbB-2 protein expression Gynecol Oncol 2002 85 469 475 12051876 10.1006/gyno.2002.6648 Leung TW Cheung AN Cheng DK Wong LC Ngan HY Expressions of c-erbB-2, epidermal growth factor receptor and pan-ras proto-oncogenes in adenocarcinoma of the cervix: correlation with clinical prognosis Oncol Rep 2001 8 1159 1164 11496335 Hove MG Dinh TV Hannigan EV Lucci JA 3rdChopra V Smith ER To T Oncogene expression and microvessel count in recurrent and nonrecurrent stage Ib squamous cell carcinoma of the cervix J Reprod Med 1999 44 493 496 10394542 Nishioka T West CM Gupta N Wilks DP Hendry JH Davidson SE Hunter RD Prognostic significance of c-erbB-2 protein expression in carcinoma of the cervix treated with radiotherapy J Cancer Res Clin Oncol 1999 125 96 100 10190316 10.1007/s004320050248 Kersemaekers AM Fleuren GJ Kenter GG Van den Broek LJ Uljee SM Hermans J Van de Vijver MJ Oncogene alterations in carcinomas of the uterine cervix: overexpression of the epidermal growth factor receptor is associated with poor prognosis Clin Cancer Res 1999 5 577 586 10100709 Scholl S Beuzeboc P Pouillart P Targeting HER2 in other tumor types Ann Oncol 2001 12 S81 87 11521727 10.1023/A:1011192622688 Bookman MA Darcy KM Clarke-Pearson D Boothby RA Horowitz IR Evaluation of monoclonal humanized anti-HER2 antibody, trastuzumab, in patients with recurrent or refractory ovarian or primary peritoneal carcinoma with overexpression of HER2: a phase II trial of the Gynecologic Oncology Group J Clin Oncol 2003 21 283 290 12525520 10.1200/JCO.2003.10.104 Haddad R Colevas AD Krane JF Cooper D Glisson B Amrein PC Weeks L Costello R Posner M Herceptin in patients with advanced or metastatic salivary gland carcinomas. A phase II study Oral Oncol 2003 39 724 727 12907212 10.1016/S1368-8375(03)00097-6 Yaziji H Goldstein LC Barry TS Werling R Hwang H Ellis GK Gralow JR Livingston RB Gown AM HER-2 testing in breast cancer using parallel tissue-based methods JAMA 2004 291 1972 1977 15113815 10.1001/jama.291.16.1972 Fujimura M Katsumata N Tsuda H Uchi N Miyazaki S Hidaka T Sakai M Saito S HER2 Is Frequently Over-expressed in Ovarian Clear Cell Adenocarcinoma: Possible Novel Treatment Modality Using Recombinant Monoclonal Antibody against HER2, Trastuzumab Jpn J Cancer Res 2002 93 1250 1257 12460467 Iwamoto H Fukasawa H Honda T Hirata S Hoshi K HER2 expression in ovarian clear cell carcinomas Int J Gynecol Cancer 2003 13 28 31 12631216 Hirsch FR Varella-Garcia M Franklin WA Veve R Chen L Helfrich B Zeng C Baron A Bunn PA Jr Evaluation of HER2 gene amplification and protein expression in non-small cell lung carcinomas Br J Cancer 2002 86 1449 1456 11986780 10.1038/sj.bjc.6600286 Nathanson DR Culliford AT 4thShia J Chen B D'Alessio M Zeng ZS Nash GM Gerald W Barany F Paty PB HER 2/neu expression and gene amplification in colon cancer Int J Cancer 2003 105 796 802 12767065 10.1002/ijc.11137 Matsuyama S Kitajima Y Sumi K Mori D Satoh T Miyazaki K Gallbladder cancers rarely overexpress HER2, demonstrated by Hercep Test Oncol Rep 2004 11 815 819 15010878 Potti A Hille R Koch M Immunohistochemical determination of HER2 in malignant melanoma Anticancer Res 2003 23 4067 4069 14666720 Bellone S Palmieri M Gokden M Joshua J Roman JJ Pecorelli S Cannon MJ Santin AD Selection of HER2-positive tumor cells in early stage cervical cancer: implications for Trastuzumab-mediated therapy Gynecol Oncol 2003 91 231 240 14529687 10.1016/S0090-8258(03)00460-8 Vogel CL Cobleigh MA Tripathy D Gutheil JC Harris LN Fehrenbacher L Slamon DJ Murphy M Novotny WF Burchmore M Shak S Stewart SJ Press M Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer J Clin Oncol 2002 20 719 726 11821453 10.1200/JCO.20.3.719 Gatzemeier U Groth G Butts C Van Zandwijk N Shepherd F Ardizzoni A Barton C Ghahramani P Hirsh V Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer Ann Oncol 2004 15 19 27 14679114 10.1093/annonc/mdh031 Gancberg D Di Leo A Cardoso F Rouas G Pedrocchi M Paesmans M Verhest A Bernard-Marty C Piccart MJ Larsimont D Comparison of HER-2 status between primary breast cancer and corresponding distant metastatic sites Ann Oncol 2002 13 1036 1043 12176781 10.1093/annonc/mdf252 Eccles SA The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis J Mammary Gland Biol Neoplasia 2001 6 393 406 12013529 10.1023/A:1014730829872 Knuefermann C Lu Y Liu B Jin W Liang K Wu L Schmidt M Mills GB Mendelsohn J Fan Z HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells Oncogene 2003 22 3205 3212 12761490 10.1038/sj.onc.1206394 Berchuck A Rodriguez G Kamel A Soper JT Clarke-Pearson DL Bast RC Jr Expression of epidermal growth factor receptor and HER2 in normal and neoplastic cervix, vulva, and vagina Obstet Gynecol 1990 76 381 387 1974342 Mark HF Feldman D Das S Sun CL Samy M Lathrop J HER-2/neu oncogene amplification in cervical cancer studied by fluorescent in situ hybridization Genet Test 1999 3 237 242 10464675 Dellas A Torhorst J Gaudenz R Mihatsch MJ Moch H DNA copy number changes in cervical adenocarcinoma Clin Cancer Res 2003 9 2985 2991 12912946 Earp HS Dawson TL Li X Yu H Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research Breast Cancer Res Treat 1995 35 115 32 7612898 Hammock L Lewis M Phillips C Cohen C Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization Hum Pathol 2003 34 1043 1047 14608539 10.1053/S0046-8177(03)00409-X

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==== Front BMC PediatrBMC Pediatrics1471-2431BioMed Central London 1471-2431-4-171534166710.1186/1471-2431-4-17Study ProtocolGlutamine-enriched enteral nutrition in very low birth weight infants. Design of a double-blind randomised controlled trial [ISRCTN73254583] van den Berg Anemone [email protected] Elburg Ruurd M [email protected] Jos WR [email protected] Willem PF [email protected] Department of Paediatrics, Division of Neonatology, VU University Medical Center, Amsterdam, the Netherlands2 Institute of Research in Extramural Medicine, VU University Medical Center, Amsterdam, the Netherlands2004 1 9 2004 4 17 17 12 8 2004 1 9 2004 Copyright © 2004 van den Berg et al; licensee BioMed Central Ltd.2004van den Berg et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. The amino acid glutamine plays an important role in maintaining functional integrity of the gut. In addition, glutamine is utilised at a high rate by cells of the immune system. In critically ill patients, glutamine is considered a conditionally essential amino acid. VLBW infants may be especially susceptible to glutamine depletion as nutritional supply of glutamine is limited in the first weeks after birth. Glutamine depletion has negative effects on functional integrity of the gut and leads to immunosuppression. This double-blind randomised controlled trial is designed to investigate the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. Furthermore, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response. Methods VLBW infants (gestational age <32 weeks and/or birth weight <1500 g) are randomly allocated to receive enteral glutamine supplementation (0.3 g/kg/day) or isonitrogenous placebo supplementation between day 3 and 30 of life. Primary outcome is time to full enteral feeding (defined as a feeding volume ≥ 120 mL/kg/day). Furthermore, incidence of serious infections and short-term outcome are evaluated. The effect of glutamine on postnatal adaptation of the gut is investigated by measuring intestinal permeability and determining faecal microflora. The role of glutamine in modulation of the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation. ==== Body Background Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. In particular, small for gestational age VLBW infants may have impaired gut function, as fetal blood flow to heart, brain and adrenals is compensatory increased, while other organs including the gastro-intestinal tract are relatively hypoperfused in intrauterine growth retardation [1]. Experimental studies have shown that the amino acid glutamine plays an important role in maintaining functional integrity of the gut [2-5]. Glutamine serves as fuel for enterocytes [2] and provides nitrogen for the synthesis of amino sugars, involved in maintenance of tight junctions [3] and mucin synthesis [4]. Moreover, glutamine has a stimulatory and regulatory effect on mucosal cell proliferation and differentiation [5]. Glutamine is not only utilised at a high rate by intestinal epithelium but also by cells of the immune system. In vitro studies have shown that increasing availability of glutamine stimulates proliferation of T-lymphocytes in response to T-cell mitogens [6], phagocytosis and antigen presentation by monocytes [7] and Th1 cytokine response [8]. In critically ill patients, endogenous glutamine synthesis cannot meet increased demand and for this reason glutamine is considered a conditionally essential amino acid [9]. VLBW infants may be especially susceptible to glutamine depletion as placental supply suddenly ceases at birth, tolerance of enteral nutrition is limited and parenteral nutrition does not contain glutamine for solubility and stability reasons. Glutamine depletion has negative effects on the functional integrity of the gut [10] and leads to immunosuppression [11]. Studies in adults have shown that glutamine supplementation decreases mortality in critically ill adults [12], infectious morbidity in recipients of bone marrow transplantation [13] and multiple trauma patients [14] and length of hospital stay in surgical patients [15]. In VLBW infants, only two studies have investigated efficacy of glutamine supplementation [16,17]. In the study of Lacey et al [16], 78 VLBW infants at high risk of developing necrotising enterocolitis (birth weight 530–1250 g) were randomised to receive either standard or glutamine-supplemented parenteral nutrition. After exclusion of 34 infants, 22 treated and 22 control infants were compared for length of stay, days on total parenteral nutrition, days on the ventilator and infectious morbidity. In infants with a birth weight ≥ 800 g no effect of glutamine supplementation was found. However, in infants with a birth weight <800 g glutamine supplementation was associated with shorter time to full enteral nutrition, fewer days on parenteral nutrition, fewer days on ventilatory support and reduced length of stay. Incidence of positive blood cultures and rate of weight gain were not different in glutamine and control groups. Neu et al [17] performed a randomised controlled trial of glutamine-enriched enteral nutrition in 68 VLBW infants with a gestational age of 24–32 weeks and a birth weight of 500–1250 g. Analysis was performed in 66 infants on an intention to treat basis. Feeding tolerance (as measured by number of days on which feeding had to be withheld) was better in the glutamine group compared to the control group. In addition, after adjusting for birth weight the odds ratio of developing sepsis was 3.8 for the control group compared to the glutamine group. Average weight at different time intervals and length of stay were not different between the groups. Although some methodological concerns can be raised (the sample size of both studies is small; Lacey et al [16] did not perform analysis on an intention to treat basis), these studies suggest that glutamine supplementation enhances feeding tolerance and decreases infectious morbidity in VLBW infants. The current double-blind randomised controlled trial is designed to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance in a sufficient large population VLBW infants. We hypothesise that time to full enteral feeding is shorter in infants who receive glutamine-enriched nutrition compared to infants in the control group. Furthermore, infectious morbidity and short-term outcome are evaluated. To elucidate the effect of glutamine-enriched enteral nutrition on the functional integrity of the gut, intestinal permeability for macromolecules is measured. As part of the postnatal adaptation of the gut, the intestinal permeability decreases during the first days of life [18]. We hypothesise that glutamine-enriched enteral nutrition stimulates postnatal adaptation of the gut, reflected by a larger decrease in intestinal permeability. Another aspect of postnatal adaptation of the gut is the development of intestinal microflora. In VLBW infants, the colonisation by bacteria (including beneficial Bifidobacterium and Lactobacillus species [19,20]) commonly present in healthy breast fed infants is delayed [21]. Intestinal mucin is an important site for bacterial adhesion and colonisation [22]. Glutamine may improve mucin quality [4] and consequently influence bacterial colonisation. We hypothesise that glutamine-enriched enteral nutrition stimulates the presence of Bifidobacterium and Lactobacillus species in the intestinal microflora. Intestinal microflora is investigated by determining faecal microflora with a culture independent technique. The effect of glutamine-enriched enteral nutrition on the immune response is investigated by determining plasma T-helper type 1 (Th1) and T-helper type 2 (Th2) cytokine concentrations following in vitro whole blood stimulation. As pregnancy is associated with skewing towards Th2 immunity [23], Th2 cytokine responses dominate the neonatal immune response [24]. Exposure to microbes stimulates Th1 cytokine responses and deviates the neonatal immune response towards balanced Th1/Th2 cytokine responses [24]. We hypothesise that glutamine-enriched enteral nutrition contributes to balanced Th1/Th2 cytokine responses by stimulating the Th1 cytokine response [8]. To assess safety of glutamine-enriched enteral nutrition, plasma amino acid profiles are determined. We hypothesise that plasma amino acid profiles in glutamine and control groups will not differ during the study period. In addition, to exclude negative effects of glutamine-enriched enteral nutrition on neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years and mental/motor development at the corrected age of 2 years are assessed. In conclusion, this double-blind randomised controlled trial aims to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. In addition, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response. Methods The study is designed as a double-blind randomised clinical trial. The national central committee on research involving human subjects and the medical ethical review board of our hospital approved the study protocol. Study population Infants with a gestational age <32 weeks and/or birth weight <1500 g admitted to the level III neonatal intensive care unit (NICU) of the VU University Medical Center, Amsterdam, are eligible for participation in the study. Written informed consent is obtained from all parents. Exclusion criteria are: major congenital or chromosomal anomalies, death <48 h after birth, transfer to another hospital <48 h after birth and admission from an extraregional hospital. Treatment allocation and blinding To balance birth weight distribution into treatment groups, each infant is stratified to one of three birth weight groups (<799 g, 800–1199 g, ≥ 1200 g) and randomly allocated to treatment <48 hours after birth. An independent researcher uses a computer-generated randomisation table based on blocks of four (provided by Nutricia Nederland BV, Zoetermeer, The Netherlands) to assign infants to treatment A or B, which correspond to batch numbers on the nutrition products. Investigators, parents, medical and nursing staff are unaware of treatment allocation. The code for the batch numbers is broken after data analysis is performed. Treatment Glutamine powder contains 82% L-glutamine and 18% glucose (nitrogen 15.5 wt/wt%; 371 kcal/100 g), whereas the isonitrogenous control powder contains 100% L-alanine (nitrogen 15.7 wt/wt%, 435 kcal/100 g). The two powders are indistinguishable by appearance, colour and smell. During the study period, glutamine and control powder are monitored for stability and microbiological contamination. Between days 3 and 30 of life, supplementation is administered in increasing doses to a maximum of 0.3 g/kg glutamine per day in the glutamine group. Initially, the supplementation dose is based on birth weight. After 2 weeks the dose is adjusted to actual weight. Two members of the nursing staff daily add supplementation to breast milk or to preterm formula (Nenatal®, Nutricia Nederland B.V., Zoetermeer, The Netherlands), according to the parents' choice. Per 100 ml, Nenatal® provides 78 kcal, 2.1 g protein (casein-whey protein ratio 40:60), 4.4 g fat and 7.5 g carbohydrate. Nenatal® does not contain free L-glutamine. When infants are transferred to other hospitals before the end of the study, the protocol is continued under supervision of the principal investigator. Nutritional support Protocol guidelines for the introduction of parenteral and enteral nutrition follow current practice at our NICU. Administration of parenteral nutrition starts at day 2 and will be advanced gradually until amino-acid intake reaches 3 g/kg/day at day 6. Parenteral nutrition is discontinued if enteral feeding reaches a volume of approximately 150 mL/kg/day. Parenteral nutrition, an all-in-one mixture provided by the hospital pharmacy, contains per 100 mL 54 kcal, 8.5 g glucose, 1.7 g amino acids and 1.7 g lipids. If necessary, glucose, amino acids and lipids are given in separate solutions. Guidelines for the introduction of enteral nutrition are as follows: 1. minimal enteral nutrition starts at day 1 (6–12 mL daily); 2. enteral nutrition is advanced either from day 3 or from day 5 in case of complications: BW <p10, GA <26 weeks, Apgar score at 5 minutes <6, umbilical artery pH <7.10 or base deficit >10 mmol/L; 3. feeding is advanced at a dose of 15–20 mL/kg/day to a maximum of 150 mL/kg/day (based on actual weight). Furthermore, guidelines for reduction/withholding of enteral feeding are: 1. enteral feeding is reduced/withheld in case of gastric residuals (> total volume of past 2 feedings), bilious residuals, emesis, ileus or necrotising enterocolitis Bell's stage ≥ II [25]; 2. when signs of feeding intolerance resolve, feeding is advanced in the volume given before reduction/withholding within 2 days. For each infant in the study a feeding schedule is proposed, based on birth weight and the guidelines as mentioned above. However, the staff of our NICU has final responsibility for the administration of parenteral nutrition and advancement of enteral feeding. Study outcome measures Study outcome measures Primary outcome of the study is time to full enteral feeding, defined as a feeding volume ≥ 120 mL/kg/day. Furthermore, other parameters of feeding tolerance, infectious morbidity, and short-term outcome are evaluated (Table 1). In addition to clinical outcome, intestinal permeability, faecal flora, plasma Th1/Th2 cytokine concentrations and plasma amino acid profiles are determined during the 30 day study period (Table 2). Table 1 Clinical outcome measures Remarks Feeding tolerance Enteral feeding >120 mL/kg/day Primary outcome Age at finishing parenteral nutrition Days of no enteral feeding during study period Necrotising enterocolitis Bell et al [25] Infectious morbidity Serious infections Number of infectious episodes Cultured micro-organisms Short-term outcome Weight z scores at birth, day 30 and at discharge Usher et al [33] Patent ductus arteriosus Ventilatory support Use of oxygen at postmenstrual age of 36 weeks Jobe et al [34] Intraventricular hemorrhage Papile et al [35] Retinopathy of prematurity Committee for ROP [36] Death Age at discharge from NICU and at discharge home ROP = retinopathy of prematurity; NICU = neonatal intensive care unit. Table 2 Study schedule < 48 h day 7 day 14 day 30 Amino acid profile x x x x Intestinal permeability x x x x Faecal flora x x x x Th1/Th2 cytokine profile x x x - Clinical outcome measures The following perinatal characteristics are registered to assess prognostic similarity: maternal age and race, obstetric diagnosis, administration of antenatal steroids and antibiotics, mode of delivery, sex, gestational age, birth weight, birth weight <p10, Apgar scores, pH of the umbilical artery, clinical risk index for babies [26] and administration of surfactant. During the study period actual intake of enteral and parenteral nutrition, powder supplementation and type of feeding (breast milk or preterm formula) are recorded daily. Evaluation of medical records for the presence of serious infections is performed by one investigator/neonatologist, unaware of treatment allocation. Serious infections include sepsis, meningitis, pyelonephritis, pneumonia, and arthritis. Sepsis work-up consists of blood, cerebrospinal fluid and urine (suprapubic bladder tap) culture. Sepsis is defined as the combination of a positive blood culture and the presence of at least two clinical signs (body temperature <36.5°C or >37.5°C, hypotension, tachycardia, apnoeic attacks, feeding problems, irritability or apathy). Meningitis is diagnosed when micro-organisms are cultured in the cerebrospinal fluid. Pyelonephritis is diagnosed when both urine culture and dimercaptosuccinic acid (DMSA) renal scan are positive. Pneumonia is defined as the combination of a positive culture of tracheal aspirate, bronchial secretion or sputum and the presence of at least one clinical sign in ventilated infants (purulent sputum, changed sputum characteristics or deterioration of ventilation settings) or at least two clinical signs in non-ventilated infants (tachypnea, cyanosis, wheezing/rales/crepitation or purulent sputum/changed sputum characteristics). Arthritis is defined as the combination of a positive culture of intra-articular fluid and the presence of signs of articular inflammation. Postnatal adaptation of the gut The effect of glutamine-enriched enteral nutrition on postnatal adaptation of the gut is studied by measuring intestinal permeability and by determining faecal flora. Intestinal permeability is measured by the sugar absorption test, as previously described [18]. After instillation of the test solution, 2 ml/kg by nasogastric tube, urine is collected for 6 hours. After collection, 0.5 ml chlorohexidine digluconate 20% (preservative) is added to the urine and samples are stored at -20°C until analysis. Lactulose and mannitol concentrations (mmol/mol creatinine) are measured by gas chromatography as previously described [27]. The lactulose/mannitol ratio is used as a measure of intestinal permeability. Faecal samples are stored at -20°C until analysis by fluorescent in situ hybridisation (FISH) using specific 16S rDNA-targeted probes as described by Harmsen et al [28]. Immune response The effect of glutamine enriched-enteral nutrition on the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation. Heparinized blood (0.5 mL), diluted 1:1 in sterile medium (RPMI 1640 without L-glutamine, Gibco, Paisley, United Kingdom) is stimulated for 24 h at 37°C in the presence of anti-CD3/anti-CD28 (Central Laboratory of the Netherlands Red Cross Blood Transfusion service, Amsterdam, the Netherlands) and Escherichia coli lipopolysaccharide (concentration 1:1000 both). After incubation, blood is centrifugated, supernatant is collected and stored at -20°C until analysis. Th1 cytokines IFN-γ, TNF-α, IL-2 and Th2 cytokines IL-4, IL-5 and IL-10 are measured by cytometric bead array (BD biosciences, Alphen aan den Rijn, the Netherlands). Safety Safety of enteral glutamine supplementation in a dosage of 0.3 g/kg/day is investigated by determination of plasma amino acid profiles. Immediately after withdrawal, heparinized blood (0.5 mL) is centrifuged at 10000 rpm for 4 minutes. Plasma is deproteinized by sulfosalicylic acid (2mg/100 μL) and stored at -70°C until analysis. Amino acid profiles are determined by high-performance liquid chromatography as described by Teerlink et al [29]. To investigate neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years [30] and mental/motor development at the corrected age of 2 years are assessed [31]. Sample size We have calculated that a sample size of 102 infants is necessary to detect a difference of at least 2.5 days in time to full enteral feeding, assuming a SD of 4.5 days (two-tailed α = 0.05, β = 0.20). The SD value is based on an retrospective analysis of time to full enteral feeding in infants with GA <32 weeks and/or BW <1500 g admitted to our NICU in 1998. Statistical analysis To determine whether randomisation is successful, prognostic similarity (perinatal and nutritional characteristics) between treatment groups is assessed. The Students' t-test, Mann-Whitney U test, and chi-square test or Fisher's exact test are used to compare continuous normally distributed data, nonparametric continuous data and dichotomous data respectively. Cox regression is performed to examine the effect of glutamine-enriched enteral nutrition on time to full enteral feeding. Logistic regression is performed to examine whether glutamine-enriched enteral nutrition influences the incidence of serious infections. In an additional analysis, adjustments are made for possible confounding factors as administration of antenatal corticosteroids, birth weight <p10, administration of breast milk and other prognostic factors that may be different between treatment groups. Analyses of secondary outcomes (only crude) is performed by Mann-Whitney U test, chi-square test or Fisher's exact test and log rank test for nonparametric continuous data, dichotomous data, and time-dependent data respectively. Generalised estimated equations for longitudinal analysis [32] are used to analyse changes over time in intestinal permeability, faecal microflora, plasma Th1/Th2 cytokine concentrations and plasma amino acid profiles. Distribution of optimal and non-optimal neuromotor development and normal and abnormal mental/motor development in glutamine and control groups is examined by logistic regression with adjustments for possible confounding factors as gestational age and birth weight. All statistical analyses are performed on an intention to treat basis. In addition, alternative per protocol analyses are performed, excluding all patients who are not treated according to protocol, defined as more than 3 consecutive days or a total of 5 days on minimal enteral feeding or without supplementation. A p value <0.05 is considered significant (two-tailed). SPSS 9.0 (SPSS Inc., Chicago, IL, USA) and STAT 7.0 (StatCorp LP, College Station, TX, USA) are used for data analysis. Competing interests Nutricia Nederland B.V. (Zoetermeer, the Netherlands) provided Nenatal®, glutamine and placebo supplementation. Authors' contributions Ruurd van Elburg and Willem Fetter formulated the research question and wrote the study protocol. Anemone van den Berg, Ruurd van Elburg and Willem Fetter contributed to the development of the protocol. Jos Twisk gave advice on data analysis. Anemone van den Berg wrote the draft for this manuscript and the other authors reviewed the manuscript. All authors approved the final version of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements We would like to thank HN Lafeber, AM van Furth and J Knol for their contribution to the study design. ==== Refs Peeters LL Sheldon RE Jones MD JrMakowski EL Meschia G Blood flow to fetal organs as a function of arterial oxygen content Am J Obstet Gynecol 1979 135 637 646 507116 Windmueller HG Spaeth AE Respiratory fuels and nitrogen metabolism in vivo in small intestine of fed rats. Quantitative importance of glutamine, glutamate, and aspartate J Biol Chem 1980 255 107 112 7350142 Panigrahi P Gewolb IH Bamford P Horvath K Role of glutamine in bacterial transcytosis and epithelial cell injury JPEN J Parenter Enteral Nutr 1997 21 75 80 9084009 Khan J Iiboshi Y Cui L Wasa M Sando K Takagi Y Alanyl-glutamine-supplemented parenteral nutrition increases luminal mucus gel and decreases permeability in the rat small intestine JPEN J Parenter Enteral Nutr 1999 23 24 31 9888414 Rhoads JM Argenzio RA Chen W Rippe RA Westwick JK Cox AD L-glutamine stimulates intestinal cell proliferation and activates mitogen-activated protein kinases Am J Physiol 1997 272 G943 G953 9176200 Parry-Billings M Evans J Calder PC Newsholme EA Does glutamine contribute to immunosuppression after major burns? Lancet 1990 336 523 525 1975037 10.1016/0140-6736(90)92083-T Spittler A Winkler S Gotzinger P Oehler R Willheim M Tempfer C Influence of glutamine on the phenotype and function of human monocytes Blood 1995 86 1564 1569 7632965 Chang WK Yang KD Shaio MF Effect of glutamine on Th1 and Th2 cytokine responses of human peripheral blood mononuclear cells Clin Immunol 1999 93 294 301 10600341 10.1006/clim.1999.4788 Lacey JM Wilmore DW Is glutamine a conditionally essential amino acid? Nutr Rev 1990 48 297 309 2080048 O'Dwyer ST Smith RJ Hwang TL Wilmore DW Maintenance of small bowel mucosa with glutamine-enriched parenteral nutrition JPEN J Parenter Enteral Nutr 1989 13 579 585 2515303 Kafkewitz D Bendich A Enzyme-induced asparagine and glutamine depletion and immune system function Am J Clin Nutr 1983 37 1025 1030 6342356 Griffiths RD Jones C Palmer TE Six-month outcome of critically ill patients given glutamine-supplemented parenteral nutrition Nutrition 1997 13 295 302 9178278 Ziegler TR Young LS Benfell K Scheltinga M Hortos K Bye R Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. A randomized, double-blind, controlled study Ann Intern Med 1992 116 821 828 1567096 Houdijk AP Rijnsburger ER Jansen J Wesdorp RI Weiss JK McCamish MA Randomised trial of glutamine-enriched enteral nutrition on infectious morbidity in patients with multiple trauma Lancet 1998 352 772 776 9737282 10.1016/S0140-6736(98)02007-8 Morlion BJ Stehle P Wachtler P Siedhoff HP Koller M Konig W Total parenteral nutrition with glutamine dipeptide after major abdominal surgery: a randomized, double-blind, controlled study Ann Surg 1998 227 302 308 9488531 10.1097/00000658-199802000-00022 Lacey JM Crouch JB Benfell K Ringer SA Wilmore CK Maguire D The effects of glutamine-supplemented parenteral nutrition in premature infants JPEN J Parenter Enteral Nutr 1996 20 74 80 8788268 Neu J Roig JC Meetze WH Veerman M Carter C Millsaps M Enteral glutamine supplementation for very low birth weight infants decreases morbidity J Pediatr 1997 131 691 699 9403648 van Elburg RM Fetter WP Bunkers CM Heymans HS Intestinal permeability in relation to birth weight and gestational and postnatal age Arch Dis Child Fetal Neonatal Ed 2003 88 F52 F55 12496227 10.1136/fn.88.1.F52 Sakata H Yoshioka H Fujita K Development of the intestinal flora in very low birth weight infants compared to normal full-term newborns Eur J Pediatr 1985 144 186 190 4043132 Hall MA Cole CB Smith SL Fuller R Rolles CJ Factors influencing the presence of faecal lactobacilli in early infancy Arch Dis Child 1990 65 185 188 2317064 Orrhage K Nord CE Factors controlling the bacterial colonization of the intestine in breastfed infants Acta Paediatr Suppl 1999 88 47 57 10569223 10.1080/080352599750029736 Mikelsaar M Mänder R Sepp E Salminen S, von Wright A Lactic acid microflora in the human microbial ecosystem and its development In Lactic Acid Bacteria: Microbiology and Functional Aspects 1998 New York: Marcel Dekker 279 342 Wegmann TG Lin H Guilbert L Mosmann TR Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon? Immunol Today 1993 14 353 356 8363725 10.1016/0167-5699(93)90235-D Holt PG Sly PD Bjorksten B Atopic versus infectious diseases in childhood: a question of balance? Pediatr Allergy Immunol 1997 8 53 58 9617774 Bell MJ Ternberg JL Feigin RD Keating JP Marshall R Barton L Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging Ann Surg 1978 187 1 7 413500 The CRIB (clinical risk index for babies) score: a tool for assessing initial neonatal risk and comparing performance of neonatal intensive care units. The International Neonatal Network Lancet 1993 342 193 198 8100927 10.1016/0140-6736(93)92296-6 van Elburg RM Uil JJ Kokke FT Mulder AM van de Broek WG Mulder CJ Repeatability of the sugar-absorption test, using lactulose and mannitol, for measuring intestinal permeability for sugars J Pediatr Gastroenterol Nutr 1995 20 184 188 7714684 Harmsen HJ Wildeboer-Veloo AC Raangs GC Wagendorp AA Klijn N Bindels JG Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods J Pediatr Gastroenterol Nutr 2000 30 61 67 10630441 10.1097/00005176-200001000-00019 Teerlink T van Leeuwen PA Houdijk A Plasma amino acids determined by liquid chromatography within 17 minutes Clin Chem 1994 40 245 249 8313601 Samsom JF de Groot L The influence of postural control on motility and hand function in a group of 'high risk' preterm infants at 1 year of age Early Hum Dev 2000 60 101 113 11121673 10.1016/S0378-3782(00)00107-9 Bayley N Bayley Scales of Infant development II 1993 2 New York, The Psychological Corporation Twisk JWR Applied longitudinal data analysis for epidemiology A practical guide 2003 Cambridge UK: Cambridge University Press Usher R McLean F Intrauterine growth of live-born Caucasian infants at sea level: standards obtained from measurements in 7 dimensions of infants born between 25 and 44 weeks of gestation J Pediatr 1969 74 901 910 5781799 Jobe AH Bancalari E Bronchopulmonary dysplasia Am J Respir Crit Care Med 2001 163 1723 1729 11401896 Papile LA Burstein J Burstein R Koffler H Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weights less than 1,500 gm J Pediatr 1978 92 529 534 305471 An international classification of retinopathy of prematurity. The Committee for the Classification of Retinopathy of Prematurity Arch Ophthalmol 1984 102 1130 1134 6547831

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==== Front BMC PediatrBMC Pediatrics1471-2431BioMed Central London 1471-2431-4-181534509910.1186/1471-2431-4-18Research ArticleLactobacillus casei strain GG in the treatment of infants with acute watery diarrhea: A randomized, double-blind, placebo controlled clinical trial [ISRCTN67363048] Salazar-Lindo Eduardo [email protected] Percy [email protected] Miguel [email protected] Elsa [email protected] R Bradley [email protected] Department of Pediatrics, Cayetano Heredia Hospital, Lima, Peru2 Department of Physics and Mathematics, Cayetano Heredia University, Lima, Peru3 Johns Hopkins Bloomberg School of Public Health, Department of International Health, Baltimore, Maryland, USA2004 2 9 2004 4 18 18 7 4 2004 2 9 2004 Copyright © 2004 Salazar-Lindo et al; licensee BioMed Central Ltd.2004Salazar-Lindo et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Adjuvant therapy to ORT with probiotic bacteria for infants with acute watery diarrhea has been under active investigation. Most studies have been done in the developed world showing benefit only for viral mild gastroenteritis. We evaluated the effect of a milk formula containing one billion (109) cfu/ml of Lactobacillus casei strain GG (LGG) upon duration and severity of diarrhea in infants in an environment with more severe acute diarrhea, where etiologic agents other than rotavirus are involved more frequently, and where mixed infections are more prevalent. Methods Male infants aged 3–36 months brought for treatment of acute watery diarrhea of less than 48 hours were eligible. After rehydration was completed with the WHO's oral rehydration solution, patients were randomly assigned to receive a milk formula either containing LGG or not. Stool volume was periodically measured using a devise suited to collect stools separate from urine. Duration of diarrhea was estimated based on stools physical characteristics. Results Eighty nine patients received the placebo milk formula and ninety received the LGG containing formula. Both groups were comparable in their baseline characteristics. Total stool output was significantly larger (p = 0.047) in the LGG group (247.8 ml/kg) than in the placebo group (195.0 ml/kg). No significant differences were found in duration of diarrhea (58.5 hours with LGG vs. 50.4 hours with placebo), rate of treatment failure (21.1% with LGG vs. 18.0% with placebo), and proportion of patients with unresolved diarrhea after 120 hours (12.2% with LGG vs. 12.5% with placebo). The rate of stools with reducing substances after 24 hours of treatment increased significantly in both groups (from 41.4% to 72.2% with LGG and from 45.9% to 68.0% with placebo). Conclusion This study did not show a positive effect of LGG on the clinical course of acute watery diarrhea. Positive beneficial effects of LGG, as had been reported elsewhere, could have been masked in our study by worsening diarrhea due to transient lactose malabsorption. Further studies with low-lactose or non-lactose conveyors of LGG are desirable. ==== Body Background Diarrheal diseases in the developing world continue to cause significant morbidity and mortality, especially when associated with malnutrition [1]. Dehydration, potassium depletion and acidosis are the main life-threatening complications of the acute losses during watery diarrhea. More than 25 years of extensive use has demonstrated that these complications could be effectively treated or prevented by oral rehydration therapy [2]. However, the oral glucose-electrolyte rehydration solution such as that recommended by WHO and Unicef neither shortens the duration of the illness nor reduces the stool loss and may cause an increase in stool volume at least during the first hours in children with acute diarrhea [3]. Optimization of the standard WHO-ORS solution, by reducing its osmolarity, has been shown to reduce diarrhea duration, total stool output, and the need for unscheduled intravenous therapy [4]. Adjuvant therapy to ORT, based on oral administration of live probiotic bacteria aimed to improve recovery of infants from acute watery diarrhea, has been under active investigation [5]. These studies, done in the developed world, have shown a benefit only for viral mild gastroenteritis. Isolauri et al have shown that oral bacterial therapy with Lactobacillus casei strain GG (LGG) reduces both the severity and duration of acute non dehydrating rotavirus enteritis [6]. Another trial in Karelia republic, LGG was shown to decrease the duration of acute diarrhea in children with viral acute diarrhea but not in those with bacterial diarrhea [7]. In a multicenter study carried out in Europe, LGG was administrated in a hypotonic oral rehydration solution to children with acute diarrhea, showing that it was safe and resulted in shorter duration of diarrhea, less chance of a protracted course, and faster discharge from the hospital [8]. Limited information is available on the potential role of LGG in infants living in the developing world, with more severe acute watery diarrhea [9], where etiologic agents other than rotavirus are involved more frequently and where mixed infections are more prevalent [10]. We conducted this study aimed to assess the effect of a milk formula containing Lactobacillus casei strain GG (LGG) on the duration and severity of diarrhea in children with acute watery diarrhea from our hospital. The study was conducted in the Rehydration Unit of Cayetano Heredia Hospital in Lima, Peru from September 1991 to June 1992. The study protocol and Consent Form were reviewed and approved by the Ethical Committee of Cayetano Heredia University in compliance with the Helsinki Declaration. Methods Study population Male infants 3–36 months of age brought for treatment because of acute diarrhea to the Rehydration Unit of Cayetano Heredia Hospital, Lima during September 1991 to June 1992 were eligible for the study if they met the following criteria: (a) a history of three or more watery stools per day for less than 48 hours, (b) no bloody stools at the moment of first examination, (c) clinical signs of dehydration, (d) no clinical features of hypovolemic shock, (e) no clinical signs of a coexisting acute systemic illness (i.e., meningitis, sepsis, pneumonia) or a recognized chronic disease such as pulmonary tuberculosis, and (f) no history of current antibiotic or antidiarrheal medication use. Patients 3–6 months old were considered for the study only if they were not on exclusive breastfeeding. Patients were not selected if their weight for age was less than 60% of the median weight for age established by the National Center for Health and Statistics (NCHS) [11]. Only patients whose parent or legal guardian was willing and able to give written informed consent were admitted to the study. Only male patients were selected in order to facilitate separate collection of stools and urine. The study size was calculated expecting a 33% reduction in duration of diarrhea and mean volume of diarrhea stool for a significance level of 0.05 and a power of 80%. Study design Infants fulfilling the admission criteria were randomly assigned to a double-blind comparison of a milk formula containing 109 cfu/ml of Lactobacillus casei strain GG (LGG) or a similar milk formula not containing LGG. Both formulas were of identical looking and tasting. Both milk formulas were provided by the manufacturer (Valio Ltd, Helsinki, Finland) as milk powder to reconstitute with water. They were given in bottles, appropriately labeled and number-coded for each child to secure the double-blind design. The actual assignment of children to their study numbers was made at the time of their enrollment into the study. Each child admitted into the study was allocated to either treatment group by restricted randomization using random permuted blocks. The codes were prepared in Finland and kept in sealed envelopes in Lima. Baseline assessment A clinical history and physical examination was completed upon admission to identify the patient, to determine the duration, type and severity of his diarrhea, to assess associated clinical features (fever, vomiting, dehydration, abdominal pain, distension), and to establish the nutritional status and previous dietary regimen of the subject. Blood for determination of serum sodium, potassium, chloride, glucose and bicarbonate, blood urea nitrogen, microhematocrit and plasma specific gravity was drawn from each patient before starting treatment. A routine urine analysis was also made. Fecal samples or rectal swabs taken at admission were transported in Cary-Blair transport medium to Cayetano Heredia University Microbiology Laboratory for primary isolation and identification of bacterial enteropathogens. A separate vial containing phosphate buffer saline was used to transport fecal specimens for rotavirus examination. Microbiologic methods Specimens were cultured for Salmonella, Shigella, E. coli and Vibrios by standard direct and enrichment enteric media. Campylobacter was selectively cultured on sheep blood agar containing Butzler's antibiotic supplement and Aeromonas on ampicillin blood agar preceded by overnight enrichment in alkaline peptone water. Five lactose fermenting colonies of E. coli isolates per patient were serotyped according to standard methods using polyvalent sera for the recognition of enteropathogenic E. coli. Detection of Rotavirus was made by ELISA method using Rotazime®. Identification of enterotoxigenic strains of E. coli was not done. Details of the microbiologic methods used in this study are described in detail elsewhere [10]. Rehydration The degree of dehydration was assessed clinically. Dehydration was corrected and then fluid balance maintained using the oral rehydration salts (ORS) in its standard WHO's recommended formulation, following the WHO Guidelines [12]. Briefly, each child was given approximately 100 ml/kg of ORS during the first four hours. The ORS was administered in frequent sips using a spoon or by nasogastric tubes if vomit or stool output rates were high. Children were not given other fluid or foods during this period. Upon completion of this period, on-going fecal losses were replaced with the same solution, on a volume to volume basis until diarrhea ceased. Administration of the study formula Once dehydration was corrected, feeding, including administration of the assigned study formula was initiated. The first dose of the assigned study formula was administered as soon as rehydration was completed and subsequent doses were given every four hours until cessation of diarrhea or for a maximum of five days. The formula was prepared immediately before administration of each dose by a specially trained nurse that was exclusively working for the study. Measuring spoons provided by the manufacturer were used and the formula powder was kept refrigerated all the time once the container was opened. Before use, the sachets with the study formulas were kept in a -20°C refrigerator. The formula powder was diluted in warm boiled water as to provide 670 Kcal per liter. The amount of formula given to each child was calculated based on the child's body weight, each child being given 150 ml/kg/day to a maximum of 1000 ml/day. On average, each serving of 100 ml will supply 1011 cfu of LGG for those receiving the formula containing LGG. Administration of the formula was by bottle and not compulsive. If the child refused to drink the formula after reasonable attempts, no extraordinary methods, like delivery via nasogastric tubing, were used; in this situation the formula was offered again four hours later. Other foods given Patients under six months of age were fed only the randomly assigned study formula to provide a daily caloric intake of at least 100 Kcal/kg (in a volume of 150 ml/kg/day). No other foods were given for these under six months patients. Older patients were fed the assigned formula ad libitum for a maximum of 1000 ml/day. Caloric requirements were completed with a blended soft baby food prepared from fresh ingredients (chicken breast, rice, carrots, squash, potatoes, and vegetable oil), by the Hospital's Nutrition Department. This soft baby food provides 1 Kcal per gram (approximately, 10% of its calories as protein, 45% as carbohydrates and 45% as fats). A total of at least 100 Kcal/kg/day (including the calories provided by the study formula) were offered to these children. Measurement of clinical outcomes All oral intake (ORS, study formula, breast milk, plain water, soft baby food) as well as urine, stool and vomitus volume were strictly measured and recorded during hospitalization. For this purpose patients were put on metabolic beds (which have a hole in the mattress were a container was placed to allow direct stool collection). A urine collection bag was fitted to avoid contamination of stools with urine and to measure the urine volume. After the first 72 hours, differential weight of dry and soiled diapers was used instead the metabolic bed to determine stool weight if the previous observed purging rate was below 5 ml/kg/hour. Blood analyses taken at admission were repeated at 24 hours. Stool examination for pH, glucose, reducing substances, leukocytes and microscopic fat were performed at admission, 24, 72 hours and at discharge. Urine specific gravity was determined every 24 hours until discharge. Criteria for stopping treatment and discharging the patient Patients were discharged from the study 24 hours after cessation of diarrhea, or at the end of five days from admission, or at the time treatment failure occurred. At discharge each patient was categorized as have completed the trial, as a treatment failure, as an unresolved diarrhea situation, or as a withdrawal. Primary outcomes • Rate of treatment failure: proportion of patients in each study group who have recurrence or continued presence of more than 5% dehydration, worsening electrolyte abnormalities, no weight gain since admission, developing of ileus or severe diarrhea defined as a purging rate in excess of 10 ml/Kg/hr in two consecutive 4-hour periods. • Rate of unresolved diarrhea after five days of treatment: proportion of patients in each study group with continuing diarrhea after five days of treatment. • Duration of diarrhea: time in hours from admission until cessation of diarrhea • Duration of hospitalization: time in hours from admission until discharge from hospital. Patients with treatment failure are excluded. • Total stool output: volume of diarrheic stools collected from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total intake of oral rehydration solution: volume of ORS taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. Secondary outcomes • Total study formula intake: volume of study formula taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total energy intake: energy from all sources (ORS, formula) taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in kilocalories per kilogram of body weight. • Total volume of vomitus: volume of vomitus collected each day from admission until cessation of vomitus or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. • Total volume of urine: volume of urine collected each day from admission until cessation of diarrhea or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. Working definitions The following definitions were used for this study: • Carbohydrate malabsorption: stool sample giving a positive reaction for reducing substances (0.75 g % or above). • Cessation of diarrhea: passage of formed stool or passage of no stool for 12 consecutive hours. • Early withdrawal: patient who (a) developed a complicating illness, (b) presented bloody diarrhea after admission and before five days of treatment, (c) was prematurely discharged under request of his parent or legal guardian, or (d) had any protocol violation or non-compliance. Statistical analysis The data, initially collected in pre-coded forms, were entered in a database organized with a relational model under Fox Pro® v. 1.02 (Dbase® compatible formats) using a data entry program with on-line checking. The data was checked again in batch mode and finally transferred to EpiInfo® for Windows® v. 3.01 (November 2003) where the tabulations and statistical analysis were performed. Letters (A or B, maintaining blindly the treatment group assignment) identified treatment groups until all statistical analysis was completed. Only after completing the analysis investigators unblinded the treatment assignment. Baseline and outcome two-way comparisons were made between the two treatment groups. A 5% level of significance for statistical tests was set in advance for all comparisons. Chi-square Yates corrected was used to compare discrete variables. One way analysis of variance was performed for continuous variables. Data from patients prematurely withdrawn from the study was included in the analysis up to the time of withdrawal (intention-to-treat analysis). Results A total of 179 patients were admitted into the study, 90 in the LGG group and 89 in the control group. Nineteen patients, eight from the LGG group and 11 from the control group, were prematurely withdrawn from the study (figure 1) because of either one of the following reasons: bloody stools within the first 24 hours after admission (11 patients); parental non-compliance (four patients); no diarrheal stools passed within the first 24 hours since admission (two patients); typical severe cholera-like diarrheal disease improperly included (one patient); and severe systemic infection present but not recognized at admission (one patient). Baseline characteristics Table 1 presents the baseline characteristics of patients upon admission. The treatment groups were comparable in age, nutritional status, and other clinical and laboratory variables. Marginal differences were found, however, in stool output during the first four hours of the rehydration phase, before administration of the study formula. The LGG group was thus comprised by slightly more severe cases. Table 2 shows stool microbiology and parasitology at admission. The distribution of enteropathogens was similar between both groups, except for rotavirus which was found more frequently in the placebo group (39.3%) than in the LGG group (24.4%) with a marginal statistical significance for the difference (p = 0.052). Table 1 Clinical and laboratory features on admission Placebo group N LGG group N P Age (months) 14.7 ± 6.4 89 14.9 ± 7.5 90 0.878 Duration of diarrhea before admission (h) 29.5 ± 14.9 89 28.8 ± 14.9 90 0.733 N° of stool motions 24 h before admission 6.6 ± 3.2 89 7.5 ± 3.9 90 0.101 N° of patients with vomitus 24 h before admission 64 (71.9%) 89 66 (73.3%) 90 0.963 N° of patients with fever 24 h before admission 39 (43.8%) 89 43 (47.8%) 90 0.703 Clinical dehydration 89 90 0.165  Mild 48 (53.9%) 36 (40.0%)  Moderate 40 (44.9%) 52 (57.8%)  Severe 1 (1.1%) 2 (2.2%) % of weight gain after rehydration 3.0 ± 2.2 89 3.1 ± 2.3 90 0.763 Fever at admission (≥ 37.5°C) 16 (18.0%) 89 14 (15.9%) 88 0.868 Weight for height* 94.8 ± 9.3 89 94.1 ± 7.6 90 0.551 Height for age* 88.9 ± 12.1 89 97.2 ± 3.7 90 0.546 Weight for age* 88.9 ± 12.1 89 88.9 ± 10.8 90 0.952 Stool output (ml/kg) first 4 h (rehydration phase) 14.4 ± 12.4 89 18.1 ± 16.1 90 0.085 Hematocrit (%) 35.0 ± 4.2 89 35.5 ± 3.8 90 0.426 Plasma specific gravity 1.028 ± 0.002 89 1.028 ± 0.002 90 0.769 Serum Na+ (mEq/l) 136.6 ± 4.7 89 136.4 ± 4.6 87 0.770 Serum K+ (mEq/l) 4.0 ± 0.7 89 4.0 ± 0.7 87 0.985 Serum Cl (mEq/l) 102.5 ± 5.2 89 102.6 ± 5.4 87 0.874 Serum total CO2 (mmol/l) 14.0 ± 3.9 88 14.0 ± 3.9 87 0.971 Blood urea nitrogen (mg/dl) 13.1 ± 10.5 70 11.4 ± 6.6 73 0.253 Blood glucose (mg/dl) 107.5 ± 33.4 88 106.5 ± 35.9 88 0.848 N° of patients with fecal leukocytes (> 20 PMN/hpf) 30 (34.5%) 87 21 (23.6%) 89 0.154 N° of patients with microscopic fat 56 (64.4%) 87 52 (58.4%) 89 0.513 Fecal pH 6.3 ± 1.1 87 6.6 ± 1.1 89 0.126 N° of patients with CHO malabsorption 39 (45.9%) 85 36 (41.4%) 87 0.659 * % of NCHS median Values are mean ± SD or n (%) Table 2 Stool microbiology and parasitology at admission Placebo group N LGG group N P Rotavirus 33 (39.3%) 84 22 (24.4%) 90 0.052 EPEC 12 (17.6%) 68 9 (13.2%) 68 0.635 Vibrio cholerae 7 (8.0%) 88 11 (12.4%) 89 0.471 Campylobacter sp. 6 (6.8%) 88 9 (10.1%) 89 0.605 Shigella sp. 9 (10.2%) 88 4 (4.5%) 89 0.241 Aeromonas sp. 2 (2.3%) 88 1 (1.1%) 89 0.992 Salmonella sp. 0 (0.0%) 88 1 (1.1%) 89 0.995 Negative microbiology 25 (36.8%) 68 32 (47.1%) 68 0.452 Cryptosporidium parvum 6 (7.0%) 86 2 (2.2%) 89 0.256 Giardia lambliae 2 (2.3%) 86 3 (3.4%) 89 0.969 Strongyloides stercolaris 1 (1.2%) 86 0 (0.0%) 89 0.986 Trichomonas sp. 0 (0.0%) 86 2 (2.2%) 89 0.492 Negative parasitology 77 (89.5%) 86 82 (92.1%) 89 0.738 Negative microbiology and parasitology 22 (35.5%) 62 30 (44.8%) 67 0.371 No patients were found positive for the following parasites: Ascaris, Enterobius, Trichuris, E. hystolytica, Ancylostoma, Necator. ETEC identification was not done Values are n (%) Stool purging reduction The mean total stool output (Table 3) was lower in the placebo group (195.0 ml/kg) than in the LGG group (247.8 ml/kg) with a marginal statistical significance for the difference (p = 0.047). The difference between means in total stool output was -52.8 ml/kg, with 95% confidence limits between -105.4 to -0.2 ml/kg. Table 3 Clinical outcomes Placebo group N LGG group N P Difference between means Discharge condition 89 90  Early withdrawal 11 (12.4%) 8 (8.9%) 0.609  Treatment failure 16 (18.0%) 19 (21.1%) 0.734  Cessation of diarrhea 51 (57.3%) 52 (57.8%) 0.931  Unresolved diarrhea 11 (12.45%) 11 (12.2%) 0.842 Mean duration of diarrhea (hours)* 50.4 ± 28.0 51 58.5 ± 30.2 52 0.157 -19.6 to 3.4 Mean duration of hospitalization (hours)** 74.7 ± 33.7 62 81.2 ± 32.6 63 0.280 -18.4 to 5.4 Total stool output (ml/kg) 195.0 ± 171.9 89 247.8 ± 180.2 90 0.047 -105.4 to -0.2 Total oral rehydration solution intake (ml/kg) 236.5 ± 195.6 89 272.8 ± 188.1 90 0.208 -93.7 to 21.1 Total study formula intake (ml/kg) 215.5 ± 136.3 89 231.5 ± 136.6 90 0.435 -56.8 to 24.8 Total energy intake (Kcal/kg)§ 220.7 ± 140.4 89 247.4 ± 143.0 90 0.209 -69.1 to 15.7 Total volume of vomitus (ml/kg) 27.6 ± 45.5 89 27.4 ± 33.9 90 0.968 -11.8 to 12.2 Total volume of urine (ml/kg) 81.5 ± 65.2 89 87.1 ± 69.4 90 0.579 -25.7 to 14.5 * For patients who ceased diarrhea before 120 hours. ** Patients with treatment failure or early withdrawals are excluded. § Calculated from calories provided by study formula, soft foods, breastmilk, and oral rehydration solution. Values are mean ± SD, n (%), or 95% confidence limits. Duration of diarrhea The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups (table 3). For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups (table 3). The difference between means in duration of diarrhea was 8.1 hours with 95% confidence limits between -19.6 hours to 3.4. Blood chemistry No patient had to be withdrawn from the study because of worsening blood chemistry abnormalities. Carbohydrate malabsorption The proportion of patients with carbohydrate malabsorption 24 hours after admission into the study was high although not different between the treatment groups (table 4). However, the rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups with respect to admission (table 4). The pH of stools at 24 hours after admission is also significantly lower in both groups compared to the pH at admission, a result consistent with worsening carbohydrate malabsorption (table 4). Table 4 Comparison of stool characteristics at admission and 24 hours after Admission N 24 h later N P Fecal pH  Placebo group 6.3 ± 1.1 87 5.9 ± 0.8 76 0.014  LGG group 6.6 ± 1.1 89 6.1 ± 0.9 74 0.004 CHO malabsorption  Placebo group 39 (45.9%) 85 51 (68.0%) 75 0.008  LGG group 36 (41.4%) 87 52 (72.2%) 72 < 0.001 Values are mean ± SD or n (%) No adverse effects due to the study formula were notice in either group during the study. Discussion Children included into this study probably represent the type of patients with diarrheal disease most frequently seen at a health service in Peru before the cholera epidemic of 1991. They were infants under two years of age, with an episode of acute watery diarrhea, presenting with signs of mild to moderate dehydration and a slight compromise of the nutritional status. Thus, we can conclude that the results of this study can be applied to most cases of acute watery non-cholera diarrhea seen in this population setting. The comparison of baseline characteristics showed no significant differences between groups. Marginal differences (not statistically significant) were observed in two variables that measure severity of illness: stool frequency 24 hours before admission and stool output during rehydration phase. Both were higher in the LGG group. Other indicators of severity, like clinical estimate of dehydration, percentage of weight gain after rehydration and proportion of patients with vomitus before admission, were similar between both groups. On the other hand, there were more cases (not statistically significant) of rotavirus in the placebo group than in the LGG group. In order to make sure that the groups were in fact comparable, we perform the statistical adjustment for these variables using multiple regression analysis and found no association with the principal outcomes (results of this analysis are not shown in the report). In more than 55% of patients an enteropathogen could be identified. This figure could be higher because studies for ETEC were not done. The most prevalent agents were rotavirus (30.7%), EPEC (11.7%), Vibrio cholerae (10.1%) and Campylobacter sp. (8.4%), with an important proportion of patients having mixed infections. These findings differ considerably from those of the Finnish clinical trial in which LGG was also tested for efficacy in reducing acute infantile diarrhea [6]. In the Finnish trial, rotavirus accounted for more than 80% of diarrheal cases, with no cases of bacterial diarrhea. A setting of greater diversity of etiologic agents and frequently co-pathogen association is perhaps more representative of developing countries [10]. The first topic to be discussed here is the high proportion of patients that did not recovered optimally during the trial, a condition that did not depend on the assigned treatment group. About 12% of patients had an unresolved diarrhea and an additional 20% were classified as treatment failures, mostly due to severe diarrhea. In two clinical trials conducted at our unit similar rates were found. For instance, in a clinical trial designed to test the clinical efficacy of a citrate-based ORS in children with acute watery diarrhea, patients who received standard therapy (WHO-ORS) had a treatment failure rate of about 24% [13]. This rate was 40% if only rotavirus associated diarrhea was considered. In another study, in which a chicken soup-based ORS was evaluated for children with similar characteristics (not published), 15.4% treatment failure rate for all subjects standard and 20% for those with rotavirus was observed. In the Finnish study, all children recovered before 5 days and no treatment failures were reported [6]. This striking difference could be explained in part by dissimilar etiologies as was mentioned above. Bacterial diarrhea and infection with more than one pathogen might produce a more severe illness. The nutritional status is also important to explain the difference. It has been well established from studies in developing countries that diarrhea lasts longer in poorly nourished children [14]. This difference seems to be important even when only slight malnutrition is present, as is the case for the patients admitted into this study. Except for total stool output (which showed a marginal statistical significance), no significant differences in the principal outcomes between groups were found. Duration of diarrhea, duration of hospitalization, and total ORS intake were not significantly different between the experimental and placebo group. The mean volume of formula (and of lactose) administered to children was the same for both groups. The confidence limits of the difference between the two groups indicate that a maximum reduction on duration of diarrhea of 6.7% attributable to the LGG formula could have been detected with the sample size of this study. These numbers indicate that the study does have a sample size sufficient to reject a type II error. No positive effects of treatment could be demonstrated as opposed to Isolauri's experience in Finland [6]. The dose factor could not explain the difference between the two studies. The dose of LGG in Isolauri's study was 2 × 1010–11 cfu per day [6]. The dose of LGG in our study was 6–8 × 1011 cfu per day, well above the threshold dose of 10 billion cfu suggested as most effective [15]. Viability of the Lactobacillus GG in the sachets while stored in Lima was not confirmed bacteriologically. There were slightly fewer cases of rotavirus in the LGG group than in the placebo group (24.4% vs. 39.3%), although the difference was not statistically significant. LGG appears to be more effective in viral than in bacterial diarrhea [8]. These facts could explain in part the lack of beneficial effect of LGG in our study. It is possible that LGG was not able to colonize the gut of our patients in the presence of bacterial pathogens. Although colonization has been documented during rotavirus diarrhea in children [16], to our knowledge this ability has not been demonstrated when other intestinal pathogens are involved. Colonization properties of LGG in patients with bacterial enteritis would deserve further studies. Our study clearly shows that the rate of carbohydrate malabsorption raised in both treatment groups from 43.6% at admission to 70.1% 24 hours later. The amount of lactose offered to patients in this study was much higher than in the usual feeding regimen at our unit (10.5 g/kg/day vs. 3.7 g/kg/day, respectively). Although to some investigators lactose intolerance is becoming a decreasing problem [17], it is clear from these data that lactose malabsorption as a factor cannot be dismissed. We had consistently found a high proportion of patients with carbohydrate malabsorption in all similar studies conducted in our unit. Thus, one possible explanation for the lack of efficacy of LGG in our patients is that worsening diarrhea due to lactose malabsorption could have masked any beneficial effect of the LGG. If this is true, then reducing the lactose content of the formula might reveal a positive effect. Another option is to deliver the LGG in suitable non-lactose containing infant food that could be given to diarrheic children as part of their treatment. Carefully conducted studies might prove this hypothesis. Conclusions Both groups were comparable in their baseline characteristics. Marginal differences were found in variables that measured severity of illness before treatment. Total stool output was slightly higher in the LGG group reaching a marginal statistical significance. No significant differences in the other principal outcome variables were found between treatment groups. The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups. For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups. The rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups. Competing interests None declared. Authors' contributions ES-L participated in the design of the study, organized and coordinated the study, participated in the statistical analysis and drafted the manuscript. PM-L carried out the clinical work, participated in the statistical analysis and the drafting of the manuscript. MC-S carried out the statistical analysis. EC-W participated in the clinical work. RBS conceived of the study, and participated in its design and drafting of the manuscript. All authors read and approved the final manuscript Figure 1 Diagram showing the flow of study subjects throughout the study Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgements The study was supported by a grant from Valio Ltd, Helsinki, Finland to the Child Health Foundation, Columbia, Maryland. ==== Refs Bern C Martines J de Zoysa I Glass RI The magnitude of the global problem of diarrhoeal disease: a ten-year update Bull World Health Organ 1992 70 705 714 1486666 Santosham M Keenan EM Tulloch J Broun D Glass R Oral rehydration therapy for diarrhea: an example of reverse transfer of technology Pediatrics 1997 100 E10 9347004 10.1542/peds.100.5.e10 el-Mougi M el-Akkad N Hendawi A Hassan M Amer A Fontaine O Pierce NF Is a low-osmolarity ORS solution more efficacious than standard WHO ORS solution? J Pediatr Gastroenterol Nutr 1994 19 83 6 7965483 Choice Study Group Multicenter, randomized, double-blind clinical trial to evaluate the efficacy and safety of a reduced osmolarity oral rehydration salts solution in children with acute watery diarrhea Pediatrics 2001 107 613 618 11335732 10.1542/peds.107.4.613 Rautanen T Isolauri E Salo E Vesikari T Management of acute diarrhoea with low osmolarity oral rehydration solutions and Lactobacillus strain GG Arch Dis Child 1998 79 157 160 9797599 Isolauri E Juntunen M Rautanen T Sillanaukee P Koivula T A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children Pediatrics 1991 88 90 97 1905394 Shornikova AV Isolauri E Burkanova L Lukovnikova S Vesikari T A trial in the Karelian Republic of oral rehydration and Lactobacillus GG for treatment of acute diarrhoea Acta Paediatr 1997 86 460 465 9183482 Guandalini S Pensabene L Zikri MA Dias JA Casali LG Hoekstra H Kolacek S Massar K Micetic-Turk D Papadopoulou A de Sousa JS Sandhu B Szajewska H Weizman Z Lactobacillus GG administered in oral rehydration solution to children with acute diarrhea: A multicenter European trial J Pediatr Gastroenterol Nutr 2000 30 54 60 10630440 10.1097/00005176-200001000-00018 Costa-Ribeiro H Ribeiro TC Mattos AP Valois SS Neri DA Almeida P Cerqueira CM Ramos E Young RJ Vanderhoof JA Limitations of probiotic therapy in acute, severe dehydrating diarrhea J Pediatr Gastroenterol Nutr 2003 36 112 5 12500005 10.1097/00005176-200301000-00021 Pazzaglia G Sack RB Salazar-Lindo E Yi A Chea E Leon-Barua R Guerrero CE Palomino J High frequency of coinfecting enteropathogens in Aeromonas – associated diarrhea of hospitalized Peruvian infants J Clin Microbiol 1991 29 1151 1156 1864933 Kuczmarski R Orden S Grummer-Strawn L Flegal K Guo S Wei R Mei Z Curtin L Roche A Johnson C CDC Growth Charts: United States Advance Data From Vital and Health Statistics of The Center For Disease Control and Prevention 2000 Number 314 A manual for the treatment of acute diarrhoea for use by physicians and other senior health workers Geneva: World Health Organization 1990 WHO/CDD/SER/80.2 Salazar-Lindo E Sack RB Chea-Woo E Leon-Barua R Kay BA Yi A Robertson AD Bicarbonate versus citrate in oral rehydration therapy in infants with watery diarrhea: a controlled clinical trial J Pediatr 1986 108 55 60 3003317 Black RE Brown KH Becker S Malnutrition is a determining factor in diarrheal duration, but not incidence, among young children in a longitudinal study in rural Bangladesh Am J Clin Nutr 1984 39 87 94 6362391 Van Niel CW Feudtner C Garrison MM Christakis DA Lactobacillus therapy for acute infectious diarrhea in children: a meta-analysis Pediatrics 2002 109 678 684 11927715 10.1542/peds.109.4.678 Juntunen M Kirjavainen PV Ouwehand AC Salminen SJ Isolauri E Adherence of probiotic bacteria to human intestinal mucus in healthy infants and during rotavirus infection Clin Diagn Lab Immunol 2001 8 293 296 11238211 10.1128/CDLI.8.2.293-296.2001 Editorial What has happened to carbohydrate intolerance following gastroenteritis? 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==== Front BMC Public HealthBMC Public Health1471-2458BioMed Central London 1471-2458-4-381532769510.1186/1471-2458-4-38Research ArticleVitamin D deficiency and causative factors in the population of Tehran Hashemipour Sima [email protected] Bagher [email protected] Hossein [email protected] Ebrahim [email protected] Mojtaba [email protected] Mohammad [email protected] Akbar [email protected] Ali Reza [email protected] Zohreh [email protected] Ali Reza Khalili [email protected] Arash [email protected] Fargol [email protected] Endocrinology & Metabolism Research Center, Tehran University of Medical Sciences, Iran2004 25 8 2004 4 38 38 15 10 2003 25 8 2004 Copyright © 2004 Hashemipour et al; licensee BioMed Central Ltd.2004Hashemipour et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background There are multiple studies in different countries regarding the prevalence of vitamin D deficiency. These studies showed high prevalence of vitamin D deficiency in Asian countries. This study tries to elucidate the prevalence of vitamin D deficiency and its influencing factors in population of Tehran. Methods 1210 subjects 20–64 years old were randomly selected. 25 (OH) D serum levels were measured. Duration of exposure to sunlight, the type of clothing and level of calcium intake and BMI were quantified based on a questionnaire. Results A high percentage of vitamin D deficiency was defined in the study population. Prevalence of severe, moderate and mild Vitamin D deficiency was 9.5%, 57.6% and 14.2% respectively. Vitamin D serum levels had no significant statistical relation with the duration of exposure to sunlight, kind of clothing and BMI. Calcium intake in the normal vitamin D group was significantly higher than the other groups (714.67 ± 330.8 mg/day vs 503.39 ± 303.1, 577.93 ± 304.9,595.84 ± 313.6). Vitamin D serum levels in young and middle aged females were significantly lower than the older group. Conclusions Vitamin D deficiency has a high prevalence in Tehran. In order to avoid complications of vitamin D deficiency, supplemental dietary intake seems essential. vitamin d deficiencycalcium intakesunlight exposure ==== Body Background Vitamin D is an essential element for establishing and maintananing bone structure. Vitamin D deficiency results in rickets and osteomalacia. Even slight vitamin D deficiency results in secondary hyperparathyroidism and increased bone resorption [1,2]. In addition, there has been increased attention to the physiologic importance of vitamin D in non-skeletal tissues [3]. Vitamin D is supplied by consumption of vitamin D-rich foods and by vitamin D synthesis in skin. Natural nutrient materials are not a sufficient source of vitamin D to supply the body requirements; therefore where there is no supplementation of foodstuffs, the main source for vitamin D is produced by UV light [4,5]. Regarding the significant role of sunlight in vitamin D synthesis, it is quite logical to suggest low prevalence of vitamin D deficiency in tropical countries. However the studies carried out in the preceding two decades have shown a high prevalence of vitamin D deficiency in tropical countries such as China, Turkey, India, Iran and Saudi Arabia [6-14]. The prevalence of vitamin D deficiency varied between 30% and 93%. However, the majority of these studies were limited to specific age and sex groups. Therefore, elucidation of vitamin D status at the community level and in different climates of a country seems essential. The present study is a part of a national project of prevention, diagnosis and treatment of osteoporosis that investigates the prevalence of vitamin D deficiency and its influencing factors in the population of Tehran. Methods 1272 healthy men and women aged 20–69 years were selected based on randomized clustered sampling from 50 blocks in Tehran. Exclusion criteria were known hepatic or renal disease, metabolic bone disease, malabsorption, sterility, oligomenorrhea, type I diabetes, hypercortisolism, malignancy, immobility for more than one-week, pregnancy, lactation, and medications influencing bone metabolism. The study protocol was approved by research ethics committee of Endocrinology & Metabolism Research Center (EMRC). Sampling was performed after taking informed consent at the beginning of 2001 in the subjects place of residence. 1210 of 1272 selected subjects participated in this study (response rate was 95%). One fasting blood sample was taken from each participant in his/her place of residence. Sample centrifuge and serum extraction were done in the field. Then samples were sent to the EMRC laboratory for analysis and were frozen immediately. 25-hydroxy vitamin D (25(OH) D) level was measured with RIA method (Biosource Europes.A,Ò). Normal range for serum vitamin D (25(OH) D) was 23 to 113 nmol/l. Serum PTH measurement was done using RIA method (Diasorin,Ò). Normal range for PTH is 13 to 54 nmol/l. Interassay and Intrassy for 25(OH) D were 8%, 6.8% and for PTH were 8.9% and 6.1% respectively. The subjects were asked to complete a questionnaire at the time of bone mineral densitometry analysis. The questionnaire included details of duration of exposure to sun light in previous month (less than 30 minutes/day; between 30 to 60 minutes/day; between 60 to 120 minutes/day; more than 120 minutes/day), sunscreen cream usage, clothing (exposure of hand and face or more than). In order to quantify the level of vitamin D and calcium consumption in the previous month, a food frequency questionnaire which was designed and standard by the Iranian Nutrition Institute was completed. Height and weight were measured at this stage. 25(OH)D equal or less than 12.5 nmol/l was considered as severe vitamin D deficiency or group 1 and vitamin D more than 12.5 nmol/l and less than 25 nmol/l was considered as moderate deficiency or group 2 [15]. PTH changes in various vitamin D serum levels were applied to detect mild vitamin D deficiency which has 25 (OH)D more than 25 nmol/l and less than or equal to 35 nmol/l. Threshold for mild vitamin D deficiency was measured by applying PTH changes in different serum levels of 25(OH) D. SPSS software (version 10) was used for data analysis. In descriptional statistics 5, 50, 95 percentiles were used. Results were expressed as mean ± SD or median. To find any significant difference between groups, X2 test Kruskal-Wallis were used. Results In order to quantify serum levels of vitamin D and other biochemical parameters, serum samples were taken from 1210 subjects (response rate was 95%). 41% of subjects were male and 59% were female. Age and sex distribution of participants are shown in table 1. In the second part of study (recall for bone mineral densitometry) for 666 subjects the questionnaire was completed. Table 1 Age and Sex distribution of participants Age(year) Total number Female Male 20–29 241 128 113 30–39 308 203 105 40–49 294 191 103 50–59 209 116 93 60 > 158 77 81 Figure 1 demonstrates vitamin D levels histogram in the study population. Total prevalence of severe, moderate and mild vitamin D deficiency was 9.5 %, 57.6% and 14.2 % respectively (Figure 2). Figure 1 Histogram of Vitamin D serum levels in study population Figure 3 demonstrates 95, 50 and 5 percentiles of vitamin D according to age and sex. Figure 3 Median, 5, and 95 percentile of Vitamin D in variable age and sex groups Serum levels of vitamin D in females above 60 years was higher than in other age groups (P < 0.001: Kruskal-Wallis test). Vitamin D serum levels in females between 20–29 years and 30–39 years was less than other age groups (P < 0.001). Median vitamin D level in females in age range of 20–29 years and above 60 years was 17 nmol/l and 39 nmol/l, respectively. Prevalence of high level of vitamin D (more than 150 nmol/l) in 60–69 years old female age group was significantly more than other age and sex group (P < 0.01). In recalling for bone densitometry, 666 returned (55% of study population) in whom the effect of influencing factors was evaluated. Table 2 shows mean BMI and daily calcium intake in different vitamin D groups. BMI was not significantly different in vitamin D groups but calcium intake in normal vitamin D group was significantly higher than other groups. Table 2 Mean BMI and daily calcium intake in variable vitamin D groups Groups Parameters Vitamin D ≤ 12.5 (nmol/l) 12.5<vitamin D ≤ 25 25<vitamin D ≤ 35 35.1<vitamin D ≤ 150 BMI (kg/m2) 27.32 ± 5.02 26.44 ± 4.52 27.66 ± 5.16 26.99 ± 4.93 Calcium intake (mg/day) 503.39 ± 303.1* 577.93 ± 304.9* 595.84 ± 313.6* 714.67 ± 330.8 *Significant difference with normal group (35.1<vitamin D ≤ 150) P < 0.05 Discussion In our study the prevalence of severe and moderate vitamin D deficiency was 9.5 % and 57.6%, respectively. Mild vitamin D deficiency had a prevalence of 14.2%. Multiple studies have been carried out about the prevalence of vitamin D deficiency but they were mostly limited to a small sample size or assessed a specific age group (especially elderly). In countries where vitamin D fortified foodstuffs are available (USA and some Scandinavian countries), prevalence of vitamin D deficiency is between 1.6–14.8% in different age groups [16-18]. In other European countries where there is no vitamin D supplementation, deficiency is more prevalent. The studies which assessed middle-aged and elderly people showed vitamin D deficiency prevalence of 14% to 59.6% in these age groups [19-22]. Vitamin D deficiency prevalence is much higher in Asian countries. Fonseca and colleagues, demonstrated vitamin D level above 10 ng/ml in only 3 saudian females out of 31 [13]. Sedrani and colleagues showed vitamin D deficiency prevalence of 44%–100% in Saudian young females with different coverage and race [9,10]. Azizi & colleagues showed vitamin D level less than 18 ng/ml in half of the study population. Vitamin D deficiency prevalence in 10–19, 20–24, 30–41 was 47.4%, 59.5%, 44.8% respectively [11]. In the present study 81.3 % of subjects had vitamin D deficiency. Most studies have shown higher prevalence of vitamin D deficiency in the elderly [15-18]. Elderly females demonstrated statistically significant higher serum levels of vitamin D compared with young and middle aged females. Parenteral vitamin D intake by elderly was the major differentiating factor between various age groups that could explain high prevalence of a high level of vitamin D in elderly females. Subjects who took vitamin D in the sampling period were excluded from the study, but those who had taken vitamin D in the preceding months were not omitted. Vitamin D has a long half-life and its frequent prescription especially in elderly women with musculo-skeletal complaints can explain differences in serum vitamin D. Regarding the essential role of sunlight in vitamin D synthesis, it is quite unexpected to see a high prevalence of vitamin D deficiency in countries such as Saudi Arabia. Different hypotheses can be made such as insufficient sun exposure, clothing habits, hyper pigmentation, air pollution, insufficient intake of vitamin D and special dietary habits [27]. Although sunlight plays an essential role in vitamin D synthesis, its' role in vitamin D deficiency of Asians is not obvious. Tehran, which is located in 36° 21''N, has a mean sun exposure of 8 hours per day [28]. In the present study sun exposure was not significantly different between subjects with vitamin D deficiency and those with normal vitamin D status. Although there is sufficient sunlight in all seasons in Saudi Arabia, Sedrani showed that half of people who had more than 30 minutes of sun exposure had vitamin D less than 8 ng/ml (20 nmol/l) [10]. Holick & colleagues showed similar rate of vitamin D synthesis in Asians as of Europeans; but Asians required greater duration of exposure [29]. Other studies showed the same degree of increase in 25 (OH) D in summer months in Asians compared with Europeans [30]. In our study, there was no difference in clothing habits of vitamin D deficient group and normal group. Sedrani showed 70% vitamin D deficiency in males compared with30 % in young females in spite of greater clothing in females [10]. Another hypothesis says that air pollution prevents enough UV exposure to skin. Insufficient vitamin D intake is another hypothesis for high prevalence of vitamin D deficiency in Asians. Insufficient dietary supplies of vitamin D in countries where foodstuffs are not supplemented, leads to generally low dietary intake of vitamin D. In the Omdahl study, daily vitamin D intake in elderly healthy women was 54 units [16]. Our study does not assess daily dietary vitamin D intake. Decreased dietary calcium level induces increased serum PTH level and increased catabolism of 25 (OH) D, therefore decreased 25(OH)D is induced by dietary calcium deficiency [15]. Average calcium intake was 660 ± 350 mg/day in this study. There was no significant difference in dietary calcium intake among the different vitamin D groups. Although consumption of phytates and animal-derived proteins was not investigated in present study, high dietary consumption of phytates and low dietary intake of animal proteins is one of the suggested hypothesis for vitamin D deficiency [24,26,27]. There are other hypotheses to explain vitamin D deficiency among Asians. Awumey et al showed higher activity level of 24-hydroxylase in fibroblasts of Indian-Americans compared with controls [31]. Therefore, increased vitamin D catabolism may cause vitamin D deficiency in Asians. In order to elucidate specific etiologies responsible for high prevalence of vitamin D deficiency in Asians further studies should be carried out. It is possible that vitamin D deficiency is induced by combination of above mentioned etiologies. In order to clarify the significance of each etiologic factor, randomized controlled trials are necessary. Conclusions Given the high prevalence of vitamin D deficiency in Iran, effective solution to overcome its consequences seems indispensable. Competing interest None declared. Figure 2 Frequency of variable Vitamin D groups Pre-publication history The pre-publication history for this paper can be accessed here: Acknowledgments The authors are thankful to Dr. Fatemeh Bandarian and Dr. Seyed Mohammad Akrami for their efforts in preparing this article and to EMRC laboratory staff for their continuous co-operation. ==== Refs Parfitt AM Gallagher JC Heaney RP Johnston CC Neer R Whedon GD Vitamin D and bone health in the elderly Am J Clin Nutr 1982 Suppl 5 1014 1031 6765068 Peacoak M Effect of calcium and vitamin D insufficiency on the skeleton Osteoporos Int 1998 Suppl 8 S45 S51 Deluca HF The Vitamin D Story: a Collaborative effort of basic science and clinical medicine FASEB 1988 2 224 236 Bouillon R Degroot L, Jameson JL, Burger HG Vitamin D: Photosynthesis, metabolism, and action to clinical applications In Endocrinology 2001 3 Philadelphia: WB Saunders 1009 1028 Bouillon R Carmeliet G Daci E Vitamin D Metabolism and action Osteoporos Int 1998 Suppl 2 S13 19 10197177 Du X Greenfield H Fraser DR Vitamin D deficiency and associated factors in adolescent girl in Beijing Am J Clin Nutr 2001 74 494 500 11566648 Alagol F Shihadeh Y Boztepe H Sunlight exposure and vitamin D in Turkish women J Endocrinol Invest 2000 23 173 177 10803475 Dawodu A Agarwal M Hossain M Hypervitaminosis D and vitamin D deficiency in exclusively brest feeding infants and their mother in summer :a justification for vitamin D supplementation of brest-feeding infants J Pediatr 2003 142 169 173 12584539 10.1067/mpd.2003.63 Sedrani SH Low 25-Hydroxy vitamin D and normal serum calcium concentrations in Saudi Arabia: Riyadh region Ann Nutr Metab 1984 28 181 185 6610383 Sedrani SH Elidrissy AW Arabi KM Sunlight and vitamin D status in normal Saudi subjects Am J Clin Nutr 1983 38 129 132 6602540 Azizi F Rais-Zadeh F Mir Said Ghazi A vitamin D deficiency in a group of Tehran Population Research In Medicine 2000 4 291 303 Taha S Dost S Sedrani S 25-hydroxy vitamin D and total calcium extra ordinarily low plasma concentrations in Saudi mothers and their neonates Pediatr Res 1984 18 739 741 6472945 Fonseca V Tongia R el-Hasmi M Exposure to sunlight and vitamin D deficiency in Saudi Arabian women Postgrad Med J 1984 60 589 591 6483701 Gowami R Gupta N Gosuwami D Prevalence and significance of low 25-Hydroxy vitamin D concentrations in healthy subjects in Dehli Am J Clin Nutr 2000 72 422 475 Lips P Vitamin D deficiency and secondary hyperparathyroidism in the elderly: Consequences for bone loss and fractures and therapeutic implications Endocr Rev 2001 22 477 501 11493580 10.1210/er.22.4.477 Omdahl JL Garry PJ Hunsaker LA Nutritional status in a healthy elderly population: vitamin D Am J Clin Nutr 1982 36 1225 1232 7148741 Kinyamu HK Gallagher JC Balhorn KE Serum vitamin D metabolites and calcium absorpation in normal young and elderly free – living women and in women living in nursing homes Am J clin Nutr 1997 65 790 797 9062531 Burnard B Sloutskis D Gianoli F Serum 25-Hydroxy vitamin D: distribution and determinants in the Swiss population Am J Clin Nutr 1992 56 537 542 1503066 Chapuy MC Preziosi P Maamer M Prevalence of vitamin D insufficiency in on adult normal population Osteopros Int 1997 7 439 443 10.1007/s001980050030 Scharla SH Scheidt Nave C Reference range of 25-Hydroxys vitamin D serum concentrations in Germany Clin Lab 1996 42 475 477 Vander Wielen RP Lowik MR Vanden Berg H Serum vitamin D concentrations among elderly people in Europe Lancet 1995 346 207 210 7616799 10.1016/S0140-6736(95)91266-5 Souberbielle JC Cormier C Kindermans C Vitamin D status and redefining serum parathyroid hormone reference range in the elderly J clin Endocrinol Metab 2001 86 3086 3090 11443171 10.1210/jc.86.7.3086 Bettica P Bevilacqua M Vago T Norbiato G High prevalence of hypovitaminosis among free living postmenopausal women referred to an osteoporos outpatient clinic in northern Italy for initial screening Osteoporosis Int 1999 9 226 229 10.1007/s001980050141 Finch PJ Ang L Colston KW Blunted Seasonal variation in serum 25-hydroxy vitamin D and increased risk of osteomalacia in vegetarian London Asians Eur J clin Nutr 1992 46 509 515 1623855 Goel KM Sweet EM Logan RW Florid and subclinical rickets among immigrant children in Glasgow Lancet 1976 1 1141 1145 58196 10.1016/S0140-6736(76)91538-5 Reinhold JG Rickets in Asian immigrants Lancet 1976 2 1132 1133 62963 10.1016/S0140-6736(76)92673-8 Smith R Asian rickets and osteomalacia Q J Med 1990 76 899 901 2236475 National statistics directory book.. Iran Statistics Center: Number in publications of statistics center 2001 3589 Lo CW Paris PW Holick MF Indian and Pakistani immigrants have the same capacity as Caucasians to produce vitamin D in response to ultra violet irradiation Am J Clin Nutr 1986 44 683 685 3766454 Hunt SP O'Riodan JL Windo J Vitamin D status in different subgroups of British Asians BMJ 1976 2 1351 1354 1000229 Awumey EM Mitra DA Hollis BW Vitamin D metabolism is altered in Asian Indians in the southern United States: a clinical research study J Clin Endocrinol Metab 1998 83 169 173 9435436 10.1210/jc.83.1.169

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==== Front BMC Pulm MedBMC Pulmonary Medicine1471-2466BioMed Central London 1471-2466-4-71533933710.1186/1471-2466-4-7Research ArticleLong acting β2 agonists for stable chronic obstructive pulmonary disease with poor reversibility: a systematic review of randomised controlled trials Husereau Don [email protected] Vijay [email protected] Michel [email protected] Shaila [email protected] Robert [email protected] Canadian Coordinating Office for Health Technology Assessment (CCOHTA), 600-865 Carling Avenue, Ottawa ON K1S 5S8, Canada2 Health Research Institute, University of Ottawa, Ottawa ON K1H 8L6, Canada2004 31 8 2004 4 7 7 16 9 2003 31 8 2004 Copyright © 2004 Husereau et al; licensee BioMed Central Ltd.2004Husereau et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The long acting β2-agonists, salmeterol and formoterol, have been recommended, by some, as first line treatment of stable chronic obstructive pulmonary disease (COPD). We reviewed evidence of efficacy and safety when compared with placebo or anticholinergic agents in patients with poorly reversible COPD. Methods After searching MEDLINE, EMBASE, HealthSTAR, BIOSIS Previews, PASCAL, ToxFile, SciSearch, the Cochrane Library, and PubMed, as well as Web sites, selected journals, reference lists, and contacting drug manufacturers, two reviewers independently screened reports of randomised controlled trials of parallel or crossover design lasting four weeks or longer and including patients with a forced expiratory volume in one second (FEV1) ≤ 75% of predicted, a ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted, and < 15% improvement from baseline FEV1 after a dose of a β2 agonist. We included trials comparing salmeterol or formoterol with placebo or with ipratropium bromide and reporting one of these outcomes: lung function; exercise capacity; quality of life scores; dyspnea; exacerbations; rescue inhaler use; incidence of tachycardia, hypokalemia, or dry mouth. Two reviewers assessed the quality of included reports using the Jadad scale and allocation concealment, and abstracted data. Results Twelve trials satisfied our inclusion criteria; eight were high quality (Jadad score >2) and four were low quality (≤ 2). The adequacy of allocation concealment was unclear in all of them. We did not perform a meta-analysis due to differences in trial design and how outcomes were reported. Two trials comparing salmeterol with ipratropium did not detect differences; one trial comparing formoterol and ipratropium described greater improvement with formoterol in morning PEFR (15.3 versus 7.1 l/min, p = 0.040). Of twelve trials comparing long acting β2 agonists with placebo, six reported no improvement in exercise capacity, eleven reported improvements in FEV1 lung function (one reported no improvement), six reported less rescue inhaler usage (one reported no difference) and five reported improved dyspnea scores (two reported no improvement). Differences in quality of life were detected in one salmeterol trial ; however, two salmeterol, and one formoterol trial reported no differences. Adverse effects of interest were not reported. Conclusion In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity. ==== Body Background Bronchodilators are the primary agents used to manage chronic obstructive pulmonary disease (COPD). They modestly improve forced expiratory volume in one second (FEV1) and reduce dynamic hyperinflation; breathlessness may lessen and exercise tolerance increase despite little improvement in spirometric measurements [1]. The bronchodilators currently available for COPD include β2 agonists (e.g., salbutamol and salmeterol), anticholinergics (e.g., ipratropium bromide) and methylxanthines (e.g., theophylline). According to the Canadian guidelines for the treatment of stable COPD [2] first line treatment consists of ipratropium, two to four doses three to four times daily, plus a short acting β2 agonist administered on an "as needed" basis. If the patient uses substantial amounts of short acting β2 agonists, or if the symptoms are greater at night than in the early morning, a long acting β2 agonist (salmeterol or formoterol) is added twice daily. However, recently some have recommended the latter as first line agents for stable COPD [3], [4-7] potentially replacing the less expensive ipratropium [4], [5-8]. Several trials have demonstrated the usefulness of salmeterol and formoterol for the management of COPD[8], [9-11]. According to a 1998 meta-analysis [12], in patients with non-reversible COPD these agents produce small increases in FEV1; however, these changes alone may not correlate highly with symptom relief [13]. The authors of the meta-analysis suggested that these drugs be prescribed only for patients who find they provide definite clinical improvement: reduced breathlessness or better exercise capacity. All three trials in the meta-analysis [10,14-16]. compared long acting β2 agonists with placebo. Since then, other studies of these agents in COPD, including comparisons with anticholinergics [8,11] have appeared in the literature. Canadian provincial drug plan managers have noted a substantial increase in the use of salmeterol and formoterol in recent years, an observation supported by data from International Medical Services Canada, which collects information on Canadian patterns of drug prescribing and estimates use: between 1997 and 2001, the use of salmeterol and formoterol for COPD increased 1,150% and 1,975%, respectively, whereas the use of ipratropium for COPD decreased by 37% [17]. In light of the new trials and the recent changes in prescribing practices, we undertook a systematic review to evaluate the efficacy and safety of long acting β2 agonists when compared with placebo or anticholinergic agents in patients with stable, poorly reversible, COPD. Methods Searching We obtained published literature and conference abstracts for this document from two separate sources: (1) search results from the CCOHTA's published health technology review "Long-acting β2-agonists for maintenance therapy of stable chronic obstructive pulmonary disease: a systematic review"; and, (2) search results from CCOHTA's ongoing clinical review on long-acting β2-agonists for maintenance treatment of stable chronic obstructive pulmonary disease in mixed population. The first search was performed on MEDLINE®, EMBASE®, HealthSTAR, BIOSIS Previews® in June 2001 using a sensitive search strategy. The second search performed in December 2002 had a more focused search strategy and included PASCAL, SciSearch and ToxFile databases in addition to MEDLINE®, EMBASE® and BIOSIS Previews® databases. As designed in the search strategy, this search captured all the studies included in CCOHTA's published review as well as some additional trials published since previous search date. Search details for both searches can be found in Appendix 2 [see Additional file 2]. Regular alerts have been established on these databases to capture new studies and are ongoing in 2004. Parallel searches were performed and updated in PubMed and the Cochrane Library. In addition, we periodically searched Web sites of clinical trial registries and health technology assessment (HTA) and related agencies. Google™ and other search engines were used to retrieve conference abstracts of major respiratory associations. We also hand searched selected journals and documents in the library of the Canadian Coordinating Office for Health Technology Assessment and the bibliographies of retrieved reports. As well, we contacted the Canadian offices of the manufacturers of salmeterol and formoterol for nonconfidential information on unpublished studies. Selection Two reviewers (D R H and V K S) worked independently on these phases of the study. Disagreements were resolved by discussion and consensus; a neutral third party (M B) was consulted when necessary. The reviewers evaluated the 504 unique citations by reviewing titles and abstracts, discarding those deemed irrelevant (e.g., case reports, review articles, and studies unrelated to the use of β2 agonists for maintenance treatment of stable COPD). They then selected all reports of randomised controlled trials (RCTs) comparing salmeterol or formoterol with placebo or an anticholinergic agent, with or without the additional use of short acting β2 agonists. No restrictions were placed on dosage, but the trials had to be of either parallel or crossover design, have lasted four weeks or longer, and have included patients that met each of the following criteria. • Non-asthmatic. • Stable COPD: no infections, exacerbations, or hospitalizations in the past month. • FEV1 ≤ 75% of predicted. • Ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted. • After a dose of a short or long acting β2 agonist < 15% improvement in FEV1. Since bronchodilators are much more efficacious in asthma than in COPD, including patients with asthma would have influenced the findings. It may be difficult to determine whether chronic airflow obstruction with relatively large responses to short acting β2 agonists represents COPD with reversibility or asthma with incomplete reversibility. A suggestive feature in the differential diagnosis of COPD is irreversible airflow limitation.[18] To better reflect this and to minimize the chance of including patients with asthma, we excluded those trials in which the average FEV1 response to a bronchodilator was greater than or equal to 15%. In addition, the trials had to have investigated one of the following outcomes. • Lung function, including FEV1 and peak expiratory flow rate (PEFR). • Exercise capacity: six minute or shuttle walking test. • Health related quality of life (QoL). • Dyspnea, including symptom diary scores. • Exacerbations of COPD. • Rescue use of salbutamol, a short acting β2 agonist. • Adverse effects, including tachycardia, hypokalemia, and dry mouth. Validity assessment The reviewers independently scored the quality of the included trial reports using a five-point scale described by Jadad [19], which assigns zero to two points each for appropriateness of randomization and double blinding and zero to one point each for reporting on withdrawals and dropouts; low scores are associated with exaggerated estimates of benefit. Concealment of allocation to treatment was also categorized as adequate, inadequate, or unclear. Data abstraction The reviewers independently recorded characteristics of the trials and patients, as well as details of the interventions and outcomes. When outcome data were available only graphically, each reviewer estimated values, and the means of the two estimates were reported. Quantitative data synthesis When possible, we calculated mean differences with 95% confidence intervals (CIs) for continuous outcomes and odds ratios (ORs) with 95% CIs for binary outcomes for individual trial data using Statistics with Confidence software [20], We used intention-to-treat data when available and otherwise end point data for patients completing the trials. Qualitative data were recorded descriptively. We had intended to do a meta-analysis, pooling data on outcomes of interest. This approach is useful when the samples of individual studies are too small for detection of an effect and when results from several trials disagree in magnitude and direction of effect [21]. However, it is only appropriate when the trials are clinically homogeneous. We found that even commonly measured outcomes, such as FEV1, could not be combined by meta-analysis because of differences in how they were reported. For example, in the six trials comparing salmeterol with placebo, FEV1 was reported as a mean change in percent predicted[16], a mean change overall[15], a mean difference between trial arms[10], no difference (without data)[22], baseline and overall FEV1 (after 24 hrs without medication)[8] and as an 0 to 12 hour area-under-the-curve (FEV1-AUC) function[23] We were not successful in obtaining more data from study authors. We also had concerns about the meta-analysis of data from trials of parallel and crossover design[24] and differences in spirometry protocols including allowable medications. Therefore, we decided on a best evidence synthesis approach [25] instead. Results Trial Flow Both reviewers agreed to tentatively accept 35 of the 58 potentially relevant reports. After further evaluation one reviewer disagreed with including 14 of the 35, which resulted in a moderate level of agreement (Kappa = 0.58; 95% CI 0.39 to 0.78). Discussion revealed that this difference related primarily to confusion surrounding interpretation of one of the criteria for eligibility, and ultimately the other reviewer agreed to reject the disputed reports. The reviewers then independently selected the same nine reports [8,10,14-16,22,23,26,27] for final inclusion. Figure 1 illustrates the study selection process. The updated search strategy identified 24 additional potentially relevant reports. Of these, four reports were independently selected based on the inclusion criteria. There were no disagreements. Appendix 1 [see Additional file 1] presents all of the 69 reports excluded with reasons. Figure 1 Flow diagram of RCT screening and selection procedure. Process through which reports were selected from those potentially relevant. RCT = randomised controlled trial; ROAD = reversible obstructive airways disease; FEV1 = forced expiratory volume in one second. Study characteristics The 13 reports were of 12 trials, all funded by manufacturers of the drugs. One report [14] describes outcomes in a subset of patients fully described in another report [10]. One reports was a conference abstract [22]; the other twelve reports were journal articles. Duplicate reports were used as a source of supplementary information. Based on the reports, eight of the trials [8,15,16,23,26-29] were of high quality (score > 2) and four[10,22,30,31] of low quality (score ≤ 2). Concealment of the allocation sequence was unclear from all of the trial reports. The reviewers agreed completely about quality. Table 1 presents details of the trials and patients. Table 1 Characteristics of included randomised, double blind, controlled trials of long acting β2 agonists in maintenance therapy for chronic obstructive pulmonary disease (COPD) First author, year of publication, design Trial quality Patients meeting inclusion criteria Interventions Outcomes investigated Notes Ulrik, 1995[16] Crossover 3 66 current smokers with FEV1 of 1–2 L (< 60% of predicted) and FEV1/FVC < 60% of predicted. FEV1of <15% or 300 ml after salbutamol Salmeterol (50 μg twice daily) or placebo for 4+4 weeks; no crossover washout. FEV1, PEFR, daytime and night-time symptom scores, rescue use of salbutamol. Two week run in. Methylxanthines, corticosteroids (short oral courses) allowed. Newman, 1996[22] (abstract) Crossover 2 42 patients with mean FEV1 of 0.93 L (35% of predicted) and no response to oral steroids. Salmeterol (100 μg twice daily) or placebo for 8+8 weeks. FEV1, FVC, six minute walk test and Borg dyspnoea assessment,[26] daytime and night-time symptom scores, rescue use of salbutamol, proportion of days unable to perform normal activity, incidence of adverse events and COPD exacerbations. Two week run in. Salbutamol rescue allowed. Grove, 1996[15] Crossover 3 29 patients with FEV1 25%–75% of predicted and 5%–15% reversibility with 200 μg of salbutamol. Salmeterol (50 μg twice daily) or placebo for 4+4 weeks; one1 week crossover washout. FEV1, FVC, TLC, RV, 6 minute walk test and exertion on Borg scale, oxygen uptake. At least one week run in. Inhaled corticosteroids, anticholinergics, oral theophylline allowed. Boyd, 1997[10] Parallel 2 674 patients with FEV1 ≤ 70% and FEV1/FVC ratio ≤ 60% of predicted and 5%–15% reversibility of FEV1 with 400 or 800 μg of salbutamol. Salmeterol (50 or 100 μg twice daily) or placebo for 16 weeks. FEV1, six minute walk test and Borg dyspnoea assessment, daytime and night-time symptom scores, rescue use of salbutamol. Two week run in. Medications other than β2 agonists allowed. Jones, 1997[14] Parallel 2 283 patients with FEV1 ≤ 70% and FEV1/FVC ratio ≤ 60% of predicted; 5%–15% reversibility of FEV1 with 400 or 800 μg of salbutamol. Salmeterol (50 or 100 μg twice daily) or placebo for 16 weeks. HRQoL with SGRQ27 and SF-36[28]. Two week run in. Medications other than β2 agonists allowed. Mahler, 1999[8] Parallel 3 145 patients with FEV1 ≤ 65% and FEV1/FVC ratio ≤ 70% of predicted; ≤ 15% reversibility of FEV1 with short acting β2agonist; grade 1 baseline severity of breathlessness. Salmeterol (42 μg twice daily) or ipratropium bromide (36 μg four times daily) or placebo for 12 weeks. FEV1 AUC, six minute walk test, daytime and night-time symptom scores, dyspnoea on BDI and TDI,[29] supplemental use of salbutamol, HRQoL on CRDQ,[30] COPD exacerbations. Run in six hours to three days. Prednisone (≤ 10 mg) or equivalent or inhaled corticosteroids allowed. Rennard, 2001[23] Parallel 3 179 patients with FEV1 ≤ 65% and FEV1/FVC ratio ≤ 70% of predicted; ≤ 12% reversibility of FEV1 with salbutamol; score ≥ 1 on MMRC five point dyspnoea scale. Salmeterol (42 μg twice daily) or ipratropium (36 μg four times daily) or placebo for 12 weeks. FEV1 and FVC AUC, dyspnoea on BDI and TDI, six minute walk test and Borg dyspnoea assessment, symptom scores, QoL on CRDQ, COPD exacerbations. Corticosteroids, inhaled and oral (< 10 mg/d), allowed. Rossi, 2002[27] Parallel 3 418 patients with FEV1 < 70% and FEV1/FVC ratio ≤ 88% of predicted; < 15% reversibility of FEV1 with short acting β2agonist; grade 1 baseline severity of breathlessness. Formoterol (12 or 24 μg twice daily) or placebo or oral slow release theophylline for 12 months. FEV1 AUC. Inhaled corticosteroids and rescue use of salbutamol allowed. Stahl, 2002[26] Parallel 3 183 patients with FEV1 < 60% and FEV1/FVC < 70% of predicted; < 12% reversibility of FEV1 after single dose of formoterol. Formoterol (18 μg twice daily) or ipratropium (80 μg three times daily) or placebo for 12 weeks. FEV1, FVC, PEFR, shuttle walking test, morning and evening symptom scores, HRQoL on SGRQ. Inhaled corticosteroids at constant doses and rescue use of short acting β2 agonists allowed. Gupta, 2002[29] Parallel 4 33 patients with FEV1 < 60 % predicted and FEV1/FVC ≤ 70%; reversibility <12 % improvement of FEV1 after 400 μg salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 8 weeks FEV1, FVC, six minute walk test, HRQoL on SF-36[28], dyspnoea on BDI, patient self-assessment, and rescure inhaler usage Two week run in period. Patients required to take beclomethasone 400 μg twice daily and ipratropium 20 μg four times daily. Mahler, 2002[30] Parallel 2 158 patients with FEV1 < 65 % predicted and FEV1/FVC ≤ 70%; reversibility <12 % improvement of FEV1 after 400 μg salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 24 weeks FEV1, morning PEF, dyspnoea on BDI and TDI; rescue salbutamol use; HRQoL on CRDQ [30]; symptoms on CBSQ Randomization stratified by reversibility. Calverly, 2003[28] Parallel 5 733 patients with FEV125–70% predicted and FEV1/FVC ≤ 70%; reversibility <10 % of predicted FEV1 after salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 52 weeks FEV1, FVC, relief medication, symptom scores, night-time awakenings, exacerbation rates, HRQoL on SGRQ Two week run in and two week follow up Hanania, 2003[31] Parallel 2 163 patients with FEV1 < 65% predicted but > 700 ml (or if ≤ 700 ml > 40 % predicted) and FEV1/FVC < 65%; reversibility < 12 % of predicted FEV1 after salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 24 weeks FEV1, morning PEF, dyspnoea on BDI and TDI; rescue salbutamol use; HRQoL on CRDQ [30]; symptoms on CBSQ, exacerbation rates (all severities) Randomization stratified by reversibility AUC = area under the curve; BDI = baseline dyspnoea index;[29] CBSQ = chronic bronchitis symptom questionnaire; [42] CRDQ = chronic respiratory disease questionnaire;[30] FEV1 = forced expiratory volume in one second; FVC = forced vital capacity; HRQoL = health related quality of life; MMRC = Modified Medical Research Council; PEFR = peak expiratory flow rate; RV = residual volume; SF-36 = Medical Outcomes Study Short Form 36;[28] SGRQ = St. George's Respiratory Questionnaire;[27] TDI = transition dyspnoea index;[29] TLC = total lung capacity. Data synthesis Comparative efficacy of long acting β2 agonists and anticholinergic agents Three trials [8,23,26] that compared long acting β2 agonists and anticholinergic agents were identified. Two 12 week trials compared salmeterol, ipratropium, and placebo [8,23].; however, only one trial [8] reported data for FEV1 and transition dyspnea index (TDI) scores for the subset of patients that met our inclusion criteria, and the data were presented graphically. No significant differences (p > 0.05) between the salmeterol and ipratropium groups were observed in the change in FEV1 from baseline, in TDI scores, or in the rescue use of salbutamol [8]. In a 12 week trial [26] formoterol produced significantly greater improvement in morning PEFR from baseline to endpoint than ipratropium (15.3 versus 7.1 l/min, p = 0.040). However, the differences between the active treatment groups were not significant (p > 0.05) for percent predicted FEV1 (13% versus 7%, p > 0.05), percent predicted FVC (8% versus 8%, p > 0.05), improvement in breathlessness score (-0.21 versus -0.29, p > 0.05), or improvement in the St. George's Respiratory Questionnaire (SGRQ) total score (0.0 versus -0.5, p > 0.05). Data on adverse effects of interest, including tachycardia, hypokalemia, and dry mouth, were not available from the reports. Comparative efficacy of long acting β2 agonists and placebo Ten trials [8,10,14-16,22,23], [28-31] had salmeterol and placebo treatment arms; the other two [26,27]. had formoterol and placebo arms. Table 2 and the following text summarize outcome data only for the patients that met our inclusion criteria. Table 2 Selected results First author FEV1 Symptom scores (lower is better) Salmeterol versus placebo Ulrik[16] No significant differences in reversibility of percent predicted FEV1 with treatment. Mean (SE): 2.7% (0.4) versus 3.4% (0.4). Significant differences in median (range) symptom scores during treatment. Daytime (scale 0–5): 1.0 (0–3.4) versus 1.8 (0.1–4.0). Night-time (scale 0–4): 0.9 (0–3.4) versus 1.6 (0.1–4.0). Newman[22] No significant differences in measurements with treatment (data not reported). Symptoms significantly reduced during salmeterol compared with placebo treatment. Scale and scores not reported. Grove[15] Significant differences one and six hours after single dose and six hours after four weeks of treatment. Mean change: 120 versus 10 ml after four weeks. Boyd[10] Significant differences in improvement with treatment. Mean difference (95% CI): for salmeterol 50 μg versus placebo 97.80 (55.6 to 139.99) ml; for salmeterol 100 μg versus placebo 117.60 (67.88 to 167.32) ml. Significant difference in distribution of median daytime and night-time symptom scores between active treatment and placebo groups (CI 0.0 to 0.0 in all cases) but not between active treatment groups. Daytime (scale 0–5): baseline, 2 in each group; from week 5, 1 in active treatment groups and 2 in placebo group. Night-time (scale 0–4): baseline, 1 in placebo and salmeterol 50 μg groups and 0 in salmeterol 100 μg group; from week 1, 0 in salmeterol 50 μg group and no change in other groups. Jones[14] (Presented QoL results for subset of patients described in Boyd[10].) Gupta[29] A mean increase in predose FEV1 of 170 ml (distibution not reported) for salmeterol vs. a mean decrease of 20 ml (distribution not reported) for placebo after 8 weeks. Both salmeterol and placebo produced significant improvemnts in BDI scores, however the magnitude of increase was greater vs. placebo (3 vs. 1); 100% patients treated with salmeterol reported decreased cough and dyspnea vs. 69% (11/16) of placebo recipients Mahler 2002 [30] A mean increase of 80 ml (95%CI 35 to 125) for salmeterol vs. mean decrease of -8 ml (95%CI: -53 to 37) for placebo. Two-hour post-dose FEV1 mean increase of 175 ml (95%CI: 116 to 234) vs. mean increase of 28 ml (95%CI: -17 to 73) Mean increase of 0.5 (SE 0.4) in TDI for salmeterol recipients and 0.4 (SE 0.3) for placebo recipients. Not clinically or statistically significant. Calverly [28] A mean increase in predose FEV1of 25 ml vs. a mean decrease of -38 ml (P < 0.05) in salmeterol and placebo recipients. Smaller difference for two-hour post-dose FEV1 (data not reported). Mean scores for cough (scale 0–3); breathlessness (scale 0 to 4); sputum production (scale 0 to 3); sputum colour (scale 0 to 4): salmeterol: cough 1.36 (SE0.03); breathlessness 1.59 (0.03); sputum production 1.30 (0.03) and colour 1.35 (0.03) vs. placebo: cough 1.44 (0.03); breathlessness 1.66 (0.03), sputum production 1.34 (0.03) and colour 1.36 (0.03). Hanania[31] A mean increase of 26 ml (95%CI: -27 to 79) for salmeterol vs. mean increase of 19 ml (95%CI: -26 to 64) for placebo. Two-hour post-dose FEV1 mean increase of 119 ml (95%CI: 70 to 168) vs. mean increase of 71 ml (95%CI: 24 to 118) The magnitude of TDI responses was less in non-reversible vs. reversible patients. (Data are not reported) Salmeterol versus ipratropium versus placebo Mahler[8] Significant differences between active treatment and placebo groups but not between active treatment groups. Peak improvements with treatment: 155, 165, and 24 ml, respectively. No significant differences in change of mean daytime symptom score with treatment. No significant differences in TDI except between ipratropium and placebo groups at week 8. After 12 weeks, mean TDI 0.35, 0.98, and 0.48, respectively. Rennard[23] Significant differences between active treatment and placebo groups but not between active treatment groups. FEV1AUC 0–12 hour responses significantly greater with salmeterol and ipratropium than with placebo (data not reported). Formoterol versus placebo Rossi[27] Significant differences in estimated difference in FEV1AUC 0–12 hour responses: between formoterol 12 μg and placebo groups, 145 ml; between formoterol 24 μg and placebo groups, 141 ml. (Individual values for treatment groups not available.) Formoterol versus ipratropium versus placebo Stahl[26] Significant differences in improvement in percent predicted FEV1between active treatment and placebo groups but not between active treatment groups: 13%, 7%, and 6%, respectively. Significant differences between active treatment and placebo groups in change from baseline in breathlessness (scored 0 to 4 morning and evening). Means: -0.21, -0.29, and 0.0, respectively. CI = confidence interval; SE = standard error. Lung function FEV1 As table 2 shows, the changes in FEV1 from baseline to endpoint differed significantly (p < 0.05) between the salmeterol and placebo groups in eight of ten trials and between the formoterol and placebo groups in two trials. FVC Five trials[15,23,26,28,31] reported on this outcome. In one 4+4 week (4 weeks then crossover then 4 additional weeks) trial [15] the increase in FVC was significantly greater with salmeterol than with placebo six hours after a single dose (200 versus 30 ml, 95%CI for difference: 40 to 290) but not after four weeks of treatment (150 versus 130 ml, 95%CI for difference: -180 to 220). In one 12 week study [23] the change in FVC was significantly greater (p < 0.001) for salmeterol (and ipratropium) than for placebo on day 1, there was no loss of response during treatment, and after four weeks the morning predose values were significantly greater in the patients treated with either active drug (data not reported). In the other 12 week trial [26] the percent predicted FVC was significantly increased by the end of formoterol treatment, compared with placebo treatment, by 8% versus -0.4% (p = 0.02). In one 52 week trial).)[28], the change in mean FVC measured 12 hours after treatment was 86 ml greater (p = 0.004) in salmeterol recipients. The difference in mean change in FVC at 52 weeks was 200 ml between groups. In an 8 week trial[29], the mean increase in FVC was 280 ml in the salmeterol group compared to a fall of 8 ml in the placebo group (p < 0.05). PEFR Three trials[16,26,28].).) reported on this outcome. In one four week trial [16] salmeterol treatment compared with placebo treatment produced a mean treatment difference in morning values of 12 l/min (238 compared with 226 l/min, 95%CI for difference: 6 to 17; p < 0.001); a statistical difference for evening values was not detected [242 (95%CI: 222 TO 262) and 237 (95% CI: 217 to 257) l/min for salmeterol and placebo, p > 0.1]. The diurnal variation was significantly lower during salmeterol treatment, at 3 (95%CI: -0.9 to 6.9) versus 11 (95%CI: 7.1 to 14.9) l/min; however, the mean treatment difference was only 7 (95%CI: 3 to 11) l/min. In the other trial, lasting 12 weeks [26], the change in morning PEFR was significantly greater by the end of formoterol (or ipratropium) treatment compared with placebo treatment: 15.3 versus -0.9 l/min (p < 0.001). In one 52 week trial).)[28], the change in mean PEF values differed significantly (p < 0.0001) for salmeterol treatment, at 257 l/min (95%CI: 253 to 261) versus placebo, at 242 l/min (95%CI: 238 to 246). Exercise capacity Results (but not always data) for six minute walk tests were reported from six trials [8,10,15,22,26,29]. None of the trials found statistically significant differences between salmeterol and placebo therapy, although one 12 week trial[8] found that at week 10 the patients receiving ipratropium walked a mean of 14 (95%CI: 0.3 to 27.7) yards farther in six minutes than those receiving placebo; there were no differences in prewalk or postwalk breathlessness between the treatment groups. The only other trial reporting data [15] found a median (interquartile range) distance in six minutes of 450 (371–491) m for placebo recipients and 425 (392–473) m for salmeterol recipients; the difference was not reported to be significant, but the patients receiving salmeterol (50 μg twice daily) perceived significantly less exertion by the end of treatment, as measured on the Borg scale [median (interquartile range) 0.5 (0–1) for salmeterol versus 1 (0–2) for placebo, p = 0.004] [32]. A 16 week trial [10] found a significant (p < 0.05) reduction in postwalk breathlessness (three or more points on the 10 point Borg scale) after eight and 16 weeks of 50 μg but not 100 μg of salmeterol twice daily, compared with placebo (OR 0.62 [95% CI 0.42 to 0.91]). Similarly, an 8+8 (8 weeks then crossover then 8 additional weeks) week trial [22] did not detect a significant (p > 0.05) difference in postwalk breathlessness between patients receiving 100 μg of salmeterol and those receiving placebo. One study comparing formoterol, ipratropium, and placebo [26] reported mean changes in walking distance from baseline to endpoint, measured with the shuttle walking test, of 19.2, 17.5 and 5.1 m, respectively; the differences were not significant (p > 0.05). Dyspnea In several trials [10,16,22,26,28-31] the patients assessed symptom severity every day, generally using ordinal scales. One 12 week trial comparing salmeterol, ipratropium, and placebo [8] measured the severity of dyspnea at baseline with a multidimensional baseline dyspnea index (BDI) and changes in severity every two weeks with a TDI [33]. As table 2 shows, some differences during treatment with an active drug as compared with placebo were significant and others were not. Rescue use of a short acting β2 agonist In five of six trials salmeterol treatment was associated with less salbutamol use than was placebo treatment [10,16,22,28,29] In one trial (4+4 weeks) [16] the median numbers (range) of daytime rescue doses were 1.7 (0–6.1) and 2.6 (0–7.9), respectively, and the median numbers of night-time doses 0 (0–4.2) and 0.3 (0–5.0). In a 52 week trial.[28], the median number of rescue inhalations per day was 2 for both salmeterol and placebo recipients, but these groups were statistically different (p = 0.028). Another trial[29] reported the mean number of doses of rescue salbutamol was significantly lower during treatment in salmeterol recipients (0.59, 95%CI: 0.30 to 0.88) versus placebo recipients (1.75, 95%CI: 1.33 to 2.17). In one 12 week trial comparing salmeterol, ipratropium, and placebo [8] no significant difference was observed in additional bronchodilator use between the placebo and active drug groups. Quality of life HRQoL was evaluated in four trials[14,26,28,29] In a salmeterol study [14] a subset of a larger patient group was asked to complete the disease-specific SGRQ [34] and the Medical Outcomes Study Short Form 36 (SF-36) [35] at baseline and after 16 weeks of treatment. The SGRQ has three components: distress due to respiratory symptoms, effects of disturbances on mobility and physical activity, and psychosocial impact of the disease; negative changes represent improvement. Data from 283 patients (95 in the placebo group and 94 in each salmeterol group) were analysed; data for others were excluded because of noncompletion of one or both questionnaires at 16 weeks or inability to meet quality control criteria or both. Salmeterol 50 μg (but not 100 μg) twice daily was associated with significantly greater improvement in mean (standard deviation) SGRQ scores from baseline to endpoint than was placebo: -6.8 (13.2) versus -1.4 (11.7) for total score and -8.0 (17.6) versus 0.0 (15.7) for impact score. No significant differences between placebo and either dose of salmeterol were observed in any of the domains of the SF-36 except for "role-emotional": these scores were significantly worse for recipients of salmeterol 100 μg than for recipients of placebo. In the 52 week study).)[28], health status was assessed with the SGRQ. The adjusted mean score was not statistically different in salmeterol recipients, at 45.2 (95%CI: 44.4 to 46.0) versus placebo recipients, at 46.3 (95%CI: 45.3 to 47.2). In an 8 week study[29] the magnitude of improvement for salmeterol versus placebo recipients rated on an SF-36 scale was significantly greater for the dimensions of "general health" (p = 0.008), "health change" (p = 0.026); physical functioning" (p = 0.008) and "vitality energy and fatigue" (p = 0.008) In the trial comparing formoterol, ipratropium, and placebo [26] HRQoL was also evaluated with the disease specific SGRQ. Of the 183 patients, 144 completed the assessment; reasons for not doing so were not reported. The changes from baseline to endpoint in total score were negligible in all three groups, at 0.0, -0.5, and 1.5, respectively. COPD exacerbations Three trials[10,22,28] reported on this outcome; only one trial).)[28] defined "COPD exacerbation" as episodes that required antibiotics or corticosteroids but not hospital admission; these occurred at a mean rate of 0.54 exacerbations/patient/year in salmeterol recipients and 0.76 exacerbations/patient/year in placebo recipients (p = 0.0003). In one 16 week trial [10] the numbers (and proportions) of patients having exacerbations among those receiving salmeterol 50 or 100 μg twice daily or placebo were 75 (33%), 91 (42%), and 98 (43%), respectively. In an 8+8 week trial [22] there were fewer exacerbations during treatment with salmeterol 100 μg twice daily than during treatment with placebo (p = 0.065); data were not presented. Adverse Effects Data on adverse effects of interest were not available from the reports. Discussion We identified thirteen reports of twelve randomized controlled trials describing the effect of administering the long acting β2-agonists, salmeterol and formoterol, to patients with poorly reversible COPD. It is not clear from the reports whether the twelve selected trials had sufficient power to detect significant differences between treatment and control groups in the various subjective and objective outcome measures. Since data were not pooled for meta-analysis, we were not able to conduct a sensitivity analysis based on the quality of trial reporting. Accordingly, we cannot comment on the possible influence of quality on the effect size of the outcome measures. Clinical heterogeneity among the trials limited assessment of the overall effect of the interventions. Since we did not perform a meta-analysis, statistical heterogeneity was not an issue. We selected reports that met our inclusion criteria, regardless of publication status, language and trial quality using a systematic research methodology; this approach has been shown to minimize potential selection and publication bias and lead to more reliable conclusions[36] We made every effort to conduct our review and report its results with the highest rigour. A potential limitation of our research is that we did not seek trials comparing long acting β2-agonists marketed outside of Canada (e.g., bambuterol) or those trials comparing long acting β2-agonists to agents other than ipratropium and placebo (e.g., short-acting β2-agonists, methylxanthines). Similarly, we excluded those trials in which the FEV1 response to a bronchodilator was not reported or greater than 15%. Thus, our results may not be generalisable to the greater population of patients who can be currently defined as having COPD[18]. We plan to include a greater number of comparators and a broader population in an upcoming analysis[37] Our results are similar to those of an earlier review [12] that identified three placebo controlled trials included in our review, but there are two important differences. In the earlier review FEV1 endpoint data from the placebo and salmeterol groups in two crossover trials [15,16] were pooled; the weighted mean differences were not significant. We preferred to analyse net improvement in FEV1 (the difference from baseline to endpoint), as we felt that it more accurately reflected the impact of maintenance therapy. In addition, no trials comparing long acting β2 agonists and ipratropium were available at the time of the earlier review. Another review has recently been published.[38] However, these authors restricted their search to MEDLINE and failed to identify a clinical trial comparing formoterol with ipratropium.[26] As a consequence, the evidence describing the use of formoterol versus ipratropium is limited to a single trial.[39] In contrast, we opted to exclude this trial after identifying two trials because roughly 40% of patients exhibited partial reversibility of FEV1 (15% to 80%) to an inhaled dose of 200 mcg salbutamol at baseline. We are in agreement with the authors' summary of the evidence surrounding salmeterol versus ipratropium. We believe our findings are in accord with current guidelines, such as the GOLD guidelines, that suggest bronchodilators should be prescribed according to individual patient responses. However, policymakers with limited health service resources need to be aware of an identifiable sub-population of patients with poorly reversible COPD for which long acting β2-agonists may result in reduced efficiency (cost-effectiveness). Our research also suggests clinical investigators of COPD trials should stratify trial participants into groups for which outcomes may consistently differ. Of the trials identified, four[23,30,31,40] used this approach. Outcome information from patients with poor reversibility was also analyzed in an abstract[41] of an excluded trial but not in the published report.[39] We were unable to ascertain sufficient details surrounding this analysis to add it to our findings. Conclusions In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity. Competing interests Donald Husereau, Vijay Shukla, Michel Boucher, and Shaila Mensinkai have no competing interests to declare. CCOHTA is an independent, nonprofit health research agency funded by the federal, provincial, and territorial governments of Canada. Robert Dales sits on the advisory committees for GlaxoSmithKline (makers of the long acting β2 agonist, salmeterol) and Boeheringer Ingelheim (makers of the anticholinergic agent, ipratropium bromide). Authors' contributions DH edited and prepared the final manuscript for publication. VS led development of the research protocol, supervised the literature review, and summarized results. DH and VS were responsible for reviewing articles, judging their relevance, assessing their quality, and extracting data. MB assisted in developing the research protocol and in conflict resolution during study selection. RD assisted in developing the research protocol and provided clinical expertise. SM designed and conducted the electronic searches and provided expertise in the area of information science. All authors either wrote sections or critically reviewed drafts of this article. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Appendix 1. A list of reports considered in this review but excluded. Click here for file Additional File 2 Appendix 2. Search strategies including databases, time horizons and subject headings/keywords used to locate trials. Click here for file Acknowledgements We thank Dorothy Rhodes, IMS, for providing drug utilization data and Ann Bolster for assistance in editing the manuscript. ==== Refs O'Donnell DE Lam M Webb KA Measurement of symptoms, lung hyperinflation, and endurance during exercise in chronic obstructive pulmonary disease Am J Respir Crit Care Med 1998 158 1557 1565 9817708 Canadian Respiratory Review Panel Guidelines for the treatment of chronic obstructive pulmonary disease (COPD) Toronto: Optimal Therapy Initiative 1998 1 Cazzola M Donner CF Long-acting beta2 agonists in the management of stable chronic obstructive pulmonary disease Drugs 2000 60 307 320 10983735 Cazzola M Matera MG Should long-acting beta2-agonists be considered an alternative first choice option for the treatment of stable COPD? Respir Med 1999 93 227 229 10464885 10.1016/S0954-6111(99)90017-3 Managing stable chronic obstructive pulmonary disease Drug Ther Bull 2001 39 81 85 11760590 New paradigms in the treatment of COPD 2001 Kirkland (PQ): Medical Education Network SEREVENT® is now approved for use as initial maintenance therapy of chronic obstructive pulmonary disease (COPD) 2002 Mississauga (ON): GlaxoSmithKline Mahler DA Donohue JF Barbee RA Goldman MD Gross NJ Wisniewski ME Yancey SW Zakes BA Richard KA Anderson WH Efficacy of salmeterol xinafoate in the treatment of COPD Chest 1999 115 957 965 10208192 10.1378/chest.115.4.957 Maesen BLP Westermann CJJ Duurkens VAM van den Bosch JMM Effects of formoterol in apparently poorly reversible chronic obstructive pulmonary disease Eur Respir J 1999 13 1103 1108 10414411 10.1034/j.1399-3003.1999.13e27.x Boyd G Morice AH Pounsford JC Siebert M Peslis N Crawford C An evaluation of salmeterol in the treatment of chronic obstructive pulmonary disease (COPD) Eur Respir J 1997 10 815 821 9150318 Wadbo M Löfdal CG Larsson K Skoogh BE Tomling G Arweström E Bengtsson T Strom K A placebo-controlled comparison of the effects of formoterol and ipratropium bromide in patients with COPD [abstract] Eur Respir J 1999 14 381S Appleton S Smith B Veale A Bara A Long-acting beta2-agonists for COPD [Cochrane Review] In The Cochrane Library 2000 Oxford: Update Software Belman MJ Botnick WC Shin JW Inhaled bronchodilators reduce dynamic hyperinflation during exercise in patients with chronic obstructive pulmonary disease Am J Respir Crit Care Med 1996 153 967 975 8630581 Jones PW Bosh TK Quality of life changes in COPD patients treated with salmeterol Am J Respir Crit Care Med 1997 155 1283 1289 9105068 Grove A Lipworth BJ Reid P Smith RP Ramage L Ingram CG Jenkins RJ Winter JH Dhillon DP Effects of regular salmeterol on lung function and exercise capacity in patients with chronic obstructive airways disease Thorax 1996 51 689 693 8882074 Ulrik CS Efficacy of inhaled salmeterol in the management of smokers with chronic obstructive pulmonary disease: a single centre randomised, double blind, placebo controlled, crossover study Thorax 1995 50 750 754 7570409 Shukla VK Husereau DR Boucher M Mensinkai S Dales R Long-acting B2-agonists for maintenance therapy of stable chronic obstructive pulmonary disease: a systematic review 2002 Ottawa: Canadian Coordinating Office for Health Technology Assessment Gold Initiative for Chron ic Obstructive Lung Disease Database Jadad AR Moore RA Carroll D Jenkinson C Reynolds DJ Gavaghan DJ McQuay HJ Assessing the quality of reports of randomized clinical trials: is blinding necessary? Control Clin Trials 1996 17 1 12 8721797 10.1016/0197-2456(95)00134-4 Altman DG Machin D Bryant TN Gardner MJ editors Statistics with confidence: confidence intervals and statistical guidelines 2000 2 [London]: BMJ Books L'Abbe KA Detsky AS O'Rourke K Meta-analysis in clinical research Ann Intern Med 1987 107 224 233 3300460 Newman AM Wiggins J Smith M Taak NK Barnes P Salmeterol xinafoate (100 mcg bd) in the treatment of chronic obstructive pulmonary airways disease (COPD) [abstract] Eur Respir J 1996 9 183S Rennard SI Anderson W ZuWallack R Broughton J Bailey W Friedman M Wisniewski M Rickard K Use of a long-acting inhaled beta2-adrenergic agonist, salmeterol xinafoate, in patients with chronic obstructive pulmonary disease Am J Respir Crit Care Med 2001 163 1087 1092 11316640 Curtin F Altman DG Elbourne D Meta-analysis combining parallel and cross-over clinical trials. I: continuous outcomes Stat Med 2002 21 2131 2144 12210629 10.1002/sim.1205 Slavin RE Best evidence synthesis: an intelligent alternative to meta-analysis J Clin Epidemiol 1995 48 9 18 7853053 10.1016/0895-4356(94)00097-A Ståhl E Wadbo M Bengtsson T Ström K Löfdahl CG Health-related quality of life, symptoms, exercise capacity and lung function during treatment for moderate to severe COPD J Drug Assess 2002 5 81 94 Rossi A Kristufek P Levine BE Bernard E Thomson MH Till D Kottakis J Della Cioppa G Comparison of the efficacy, tolerability and safety of formoterol dry powder and oral slow-release theophylline in the treatment of COPD Chest 2002 121 1058 1069 11948033 10.1378/chest.121.4.1058 Calverley P Pauwels R Vestbo J Jones P Pride N Gulsvik A Anderson J Maden C Combined salmeterol and fluticasone in the treatment of chronic obstructive pulmonary disease: a randomised controlled trial Lancet 2003 361 449 456 12583942 10.1016/S0140-6736(03)12459-2 Gupta RK Chhabra SK An evaluation of salmeterol in the treatment of chronic obstructive pulmonary diseases Indian J Chest Dis Allied Sci 2002 44 165 172 12206475 Mahler DA Wire P Horstman D Chang CN Yates J Fischer T Shah T Effectiveness of fluticasone propionate and salmeterol combination delivered via the diskus device in the treatment of chronic obstructive pulmonary disease Am J Respir Crit Care Med 2002 166 1084 1091 12379552 10.1164/rccm.2112055 Hanania NA Darken P Horstman D Reisner C Lee B Davis S Shah T The efficacy and safety of fluticasone propionate (250 microg)/salmeterol (50 microg) combined in the Diskus inhaler for the treatment of COPD Chest 2003 124 834 843 12970006 10.1378/chest.124.3.834 Borg GAV Psychophysical bases of perceived exertion Med Sci Sports Exerc 1982 14 377 381 7154893 Mahler DA Weinberg DH Wells CK Feinstein AR The measurement of dyspnea: contents, interobserver agreement, and physiologic correlates of two new clinical indexes Chest 1984 85 751 758 6723384 Jones PW Quirk FH Baveystock CM Littlejohns P A self-complete measure of health status for chronic airflow limitation. The St. George's Respiratory Questionnaire Am Rev Respir Dis 1992 145 1321 1327 1595997 Ware JE Sherbourne CA Davies AR The MOS short-form general health survey 1988 Santa Monica (CA): Rand Corporation McAuley L Pham B Tugwell P Moher D Does the inclusion of grey literature influence estimates of intervention effectiveness reported in meta-analyses? Lancet 2000 356 1228 1231 11072941 10.1016/S0140-6736(00)02786-0 Shukla VK Husereau DR Boucher M Mensinkai S Dales R Long-acting B2-agonists for maintenance treatment of stable chronic obstructive pulmonary disease: a systematic review of clinical studies Ottawa: Canadian Coordinating Office for Health Technology Assessment 2004 Dougherty JA Didur BL Aboussouan LS Long-acting inhaled beta 2-agonists for stable COPD Ann Pharmacother 2003 37 1247 1255 12921509 10.1345/aph.1C436 Dahl R Greefhorst LA Nowak D Nonikov V Byrne AM Thomson MH Till D Della Cioppa G Inhaled formoterol dry powder versus ipratropium bromide in chronic obstructive pulmonary disease Am J Respir Crit Care Med 2001 164 778 784 11549532 Mahler DA Wong E Giessel G Clifford D Chang C Yates J Horstman D Fischer T Wire P Improvements in FEV1 and symptoms in COPD patients following 24 weeks of twice daily treatment with salmeterol 50/ fluticasone propionate 500 combination [abstract] Am J Respir Crit Care Med 2001 163 A279 Dahl R Greefhorst AP Thomson MH Till D Formoterol (foradil®) improves lung function and quality of life (QOL) parameters in patients with reversible or poorly reversible COPD [abstract] Am J Respir Crit Care Med 2001 163 A280 Petty TL The National Mucolytic Study. Results of a randomized, double-blind, placebo-controlled study of iodinated glycerol in chronic obstructive bronchitis Chest 1990 97 75 83 2403903

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==== Front BMC DermatolBMC Dermatology1471-5945BioMed Central London 1471-5945-4-101533313510.1186/1471-5945-4-10Research ArticleEnhanced diagnostic immunofluorescence using biopsies transported in saline Vodegel Robert M [email protected] Jong Marcelus CJM [email protected] Hillegonda J [email protected] Marijn B [email protected] Hendri H [email protected] Marcel F [email protected] Centre for Blistering Diseases, Department of Dermatology, Groningen University Hospital, Groningen, the Netherlands2004 27 8 2004 4 10 10 17 5 2004 27 8 2004 Copyright © 2004 Vodegel et al; licensee BioMed Central Ltd.2004Vodegel et al; licensee BioMed Central Ltd.This is an open-access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates. Methods In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours. Results Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25). Conclusions We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis. ==== Body Background The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases[1]. Reliable diagnosis by DIF not only requires an experienced observer, but first of all proper skin (or mucosal) biopsies with well-preserved immunoreactants. For the latter purpose biopsies are usually snap-frozen in liquid nitrogen or, alternatively, placed in Michel's fixative that facilitates transport of biopsies from outside hospitals [2-6]. But even representative and optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate desired specific staining (DSS) of the epidermal basement membrane zone (BMZ) is involved [7]. In such cases, specific IgG fluorescence is easily masked by the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates. The background fluorescence, consisting of both undesired specific staining (USS) and non-specific staining (NSS), largely determines the signal-to noise ratio [7]. This ratio in turn determines the detection threshold and thereby the diagnostic sensitivity of the DIF technique. A low ratio for IgG resulting from weak DSS and high USS plus NSS will undoubtedly yield false negative cases. So far, the signal- to noise ratio in diagnostic IF has received little attention. The present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel's fixative and normal saline. Methods Patients The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin [8,9], immunoblotting [10], and ELISA (desmoglein 1 and 3) [11]. The patients had one of the following diagnoses: bullous pemphigoid (BP; n = 5); mucous membrane pemphigoid with skin involvement (MMP; 1); linear IgA dermatosis (LAD; 1), anti-epiligrin cicatricial pemphigoid (AECP; 1); epidermolysis bullosa acquisita (EBA; 1); dermatitis herpetiformis (DH; 1); pemphigus vulgaris (PV; 3); pemphigus foliaceus (PF; 3); subacute and systemic lupus erythematosus (LE; 5); and small vessel IgA vasculitis (4). Skin biopsies and processing From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cm2 to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline. Freezing Biopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below). Fixative Michel's fixative and buffer solution were prepared monthly according to the original description.1 Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing. Saline We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF. Direct immunofluorescence microscopy For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion (PAP-pen; DAKO; Glostrup). The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab')2 antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin (DAKO; Glostrup). Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days. The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scored12;13.: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone (dermo-epidermal junction), and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores. Statistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test. Results A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel's fixative. Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies (p < 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls (Fig. 1). In comparison, fresh-frozen and fixed biopsies showed a relatively poor signal- to noise ratio for IgG (and IgA). In these biopsies, weak (+) specific fluorescence of IgG at the BMZ or IgA in vessel walls was found to be masked easily by relatively high background staining. Biopsies kept for 48 hours in saline showed a variable degree of diminution of specific staining, and tended to become negative in case of weak (+) specific fluorescence. The IgG fluorescence of epidermal in vivo ANA, present in fresh-frozen and fixed biopsies of two cases with (S)LE, became negative in saline biopsies (both 24 h and 48 h). In general, biopsies with moderate to bright (++/+++) specific fluorescence remained positive in all transport media. The reduced background fluorescence in saline biopsies occasionally revealed doubtful to weak focal IgG fluorescence of the BMZ that was regarded as non-relevant. Furthermore, we observed dermo-epidermal split formation in saline biopsies, not present in matched fresh-frozen and fixed biopsies. The extent of split formation varied among saline biopsies and increased with time (48 h > 24 h). In cases with pemphigoid, IgG was found predominantly at the epidermal side of splits, in contrast with e.g. SLE where IgG was found at the dermal side (Fig. 2). In case an artificial split is induced because of saline incubation a BMZ signal stand more out because of the dark background of the blister cavity (Fig. 2). The overall morphology in saline biopsies was quite fair and largely sufficient for the purpose of diagnostic IF microscopy. Figure 1 Comparison of direct immunofluorescence in cryosections of matched skin biopsies transported in liquid nitrogen, Michel's fixative or saline. Note the substantially reduced background fluorescence in saline-transported biopsies. Pemphigus foliaceus showing characteristic IgG fluorescence at the epidermal intercellular space. Additional granular IgG staining at the basement membrane zone (arrow) stands out most clearly in saline transported biopsy. (obj. ×20) Mucous membrane pemphigoid with skin involvement showing weak linear IgG fluorescence at the basement membrane zone that is only visible in the saline transported biopsy (arrow). (obj. ×20) Lupus erythematosus showing granular IgG fluorescence at the dermo-epidermal junction. Additional IgG staining of subepidermal vessel walls is best visible in the saline-transported biopsy. (obj. ×20) Vasculitis showing fine-granular IgA fluorescence in subepidermal capillary walls (arrows) which is most distinct in the saline-transported biopsy. (obj. ×40) Figure 2 Direct immunofluorescence (IgG, combined with transmitted light) in saline transported skin specimen of lupus erythematosus. After 48 hours in saline there is subepidermal split formation, not present in fresh-frozen (N2) and fixed (Mi48) skin. Note the still obvious granular IgG fluorescence at the dermal side of the split. (obj. ×40) The diagnostic results of direct IF in matched biopsies are summarized in Table I. By interpreting these data it should be realized that the minority of cases (20%) showed bright (+++) specific fluorescence in standard frozen biopsies, whereas the majority (52%) showed only weak to moderate (+/++) specific fluorescence of the relevant target structure(s). Two originally positive cases, one pemphigus and one IgA vasculitis became doubtful or negative (non-diagnostic) in all transport media. The highest rate of conclusive cases by direct IF was obtained in biopsies kept in saline for 24 hours (S24, 84%), and the lowest in fixed biopsies (Mi48, 50%). The case of mucous membrane pemphigoid with weak IgG (+) and IgA (+) fluorescence at the BMZ was obvious in saline biopsies (S24, S48) but non-diagnostic in matched fresh-frozen and fixed biopsies. The highest non-diagnostic percentage was obtained in fixed biopsies (32%) and the lowest in S24 saline biopsies (8%). In one case, classified as misdiagnosed, the fresh-frozen biopsy led to the diagnosis of LAD as IgA (+) and C3c (+) were the only immunoreactants observed, whereas the matched fixed and saline biopsies showed additional linear IgG (+/++) fluorescence at the BMZ (suggestive of mixed IgG/IgA pemphigoid). After correction for the missing biopsies, the results were only statistically significant comparing Mi and S24 (p < 0.05). Other comparisons (N2 versus Mi, S24 and S48 or Mi versus S48 or S24 versus S48) were not significant. Table 1 Results of direct immunofluorescence (DIF) of matched skin biopsies transported in different media. N2 Mi48 S24 S48 Diagnosis by DIF DSS n = 25 n = 22§ n = 25 n = 23§ a. conclusive +/+++ 14 (56%) 11 (50%) 21 (84%) 16 (70%) b. tentative ±/+ 5 (20%) 4 (18%) 2 (8%) 3 (13%) c. non-diagnostic -/± 5 (20%) 7 (32%) 2 (8%) 4 (17%) d. mis-diagnosed 1 (4%) - - - N2, snap-frozen specimens in liquid nitrogen; Mi48, specimens in Michel's fixative for 48 hours; S24 and S48, specimens in saline for 24 and 48 hours respectively; DSS, desired specific staining; n, number of cases § Two S48 and three Mi48 matched biopsies were either lacking or considered unsuitable. Discussion Perilesional skin biopsies kept in saline for 24 hours yielded a higher diagnostic rate in direct IF than fresh-frozen biopsies in liquid nitrogen or biopsies kept in Michel's fixative. Several authors have described the positive effect of saline in increasing the sensitivity of IF analysis. Judd and Lever showed that skin biopsies stored for 24 hours in 0.15 M phosphate buffered saline prior to freezing gave a very high incidence of positive readings in direct IF [14]. Similarly, the use of 1.0 M NaCl split skin as a diagnostic tool for direct and indirect IF has been reported to increase the sensitivity of these methods [9,15-17]. It has been suggested that the increase of IF sensitivity by saline incubation is due to improved exposure of epitopes and/or by a decrease of background staining [16]. Our data suggest that the improved DIF sensitivity in saline biopsies is primarily due to decreased background staining resulting in better image contrast (signal-to noise ratio). We know that our visual perception is sensitive for light contrast and not for intensity [18]. The staining intensity perceived of a given specific fluorescent signal is strongly correlated with the intensity of the background (compare looking at the stars in daylight and at night; the intensity is the same, but seems multiplied at night). In this respect, IgG (and IgA) fluorescence in skin tissue is hampered by relatively strong background fluorescence in the dermis that may mask weak specific fluorescence at the BMZ and in vascular walls. Saline appears to reduce this background staining, resulting in relative increase of desired specific staining and thereby enhanced diagnostic sensitivity. A disadvantage of saline is the limited time of transport (24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel's fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results [4]. The second disadvantage of saline is the loss of at least some epidermal in vivo ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration [19], and splitting at the dermo-epidermal junction, not found with Michel's fixative [6]. That is why saline is not suitable for antigen mapping in the diagnosis of genetic diseases [20]. Neither are biopsies kept in saline suited for immuno-electron microscopy [5,6,20]. However, for the diagnosis of autoimmune and immune complex-mediated diseases by DIF, it is not optimal morphology that counts, but the low detection threshold of immunoreactants. In this regard, (artificial) dermo-epidermal split-formation may add value to the method, rather than being a problem, by darkening of the juxtaposed background and mapping of the linear epidermal BMZ deposition [21]. Besides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel's fixative for further transport. Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and (optional) storage at -80°C the next day. Conclusions The use of normal saline offers an attractive alternative to liquid nitrogen and Michel's fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy. Competing interests None declared. Abbreviations BMZ: basement membrane zone BP : bullous pemphigoid DH: dermatitis herpetiformis DIF: direct immunofluorescence microscopy DSS: desired specific staining EBA: epidermolysis bullosa acquisita IF: immunofluorescence microscopy LAD: linear IgA dermatosis LE : lupus erythematosus MMP: mucous membrane pemphigoid NSS: non-specific staining PF : pemphigus foliaceus PV : pemphigus vulgaris USS: undesired specific staining Authors' contributions RV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: ==== Refs Morrison LH Direct immunofluorescence microscopy in the diagnosis of autoimmune bullous dermatoses Clin Dermatol 2001 19 607 613 11604308 10.1016/S0738-081X(00)00179-6 Michel B Milner Y David K Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases – preliminary report J Invest Dermatol 1972 59 449 452 4643885 10.1111/1523-1747.ep12627611 Nisengard RJ Blaszczyk M Chorzelski T Beutner Immunofluorescence of biopsy specimens: comparison of methods of transportation Arch Dermatol 1978 114 1329 1332 356740 10.1001/archderm.114.9.1329 Skeete MV Black MM The evaluation of a special liquid fixative for direct immunofluorescence Clin Exp Dermatol 1977 2 49 56 324665 Vaughan Jones SA Palmer I Bhogal BS Eady RAJ Black MM The use of Michel's transport medium for immunofluorerscence and immunoelectron microscopy in autoimmune bullous diseases J Cutan Pathol 1995 22 365 370 7499578 Woollons A Holmes GJ Gratian MJ Bhogal BS Black MM Michel's medium: a potential alternative to cryoprotection for tissue transport in the investigation of genetic skin disease Clin Exp Dermatol 1999 24 487 489 10606956 10.1046/j.1365-2230.1999.00540.x Beutner EH Defined immunofluorescent staining: past progress, present status, and future prospects for defined conjugates Ann N Y Acad Sci 1971 177 506 526 4110298 Woodley DT Sauder D Talley MJ Silver M Grotendorst G Qwarnstrom E Localization of basement membrane components after dermal-epiderma junction separation J Invest Dermatol 1983 81 149 153 6348179 10.1111/1523-1747.ep12543517 Gammon WR Briggaman RA Inman AO IIIQueen LL Wheller CE Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin J Invest Dermatol 1984 82 139 144 6363567 10.1111/1523-1747.ep12259692 Pas HH Immunoblot assay in differential diagnosis of autoimmune blistering skin diseases Clin Dermatol 2001 19 622 630 11604310 10.1016/S0738-081X(00)00176-0 Ishii K Amagai M Hall RP Hashimoto T Takayanagi A Gamou S Shimizu N Nishikawa T Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins J Immunol 1997 159 2010 2017 9257868 Gately LE IIINesbitt LT Jr Update on immunofluorescent testing in bullous diseases and lupus erythematosus Dermatol Clin 1994 12 133 142 8143377 10.1016/0738-081X(94)90264-X Velthuis PJ Kater L van der Tweel I Meyling FG Derksen RH Hene RJ van Geutselaar JA Baart de la Faille H In vivo antinuclear antibody of the skin: diagnostic significance and association with selective antinuclear antibodies Ann Rheum Dis 1990 49 163 167 2181945 Judd KP Lever WF Correlation of antibodies in skin and serum with disease severity in pemphigus Arch Dermatol 1979 115 428 432 373639 10.1001/archderm.115.4.428 Kelly SE Wojnarowska F The use of chemically split tissue in the detection of circulating anti-basement membrane zone antibodies in bullous pemphigoid and cicatricial pemphigoid Br J Dermatol 1988 118 31 40 3277659 Gammon WR Kowalewski C Chorzelski TP Kumar V Briggaman RA Beutner EH Direct immunofluorescence studies of sodium chloride-seprated skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita J Am Acad Dermatol 1990 22 664 670 2180996 Domloge-Hultsch N Bisalbutra P Gammon WR Yancey KB Direct immunofluorescence microscopy of 1 mol/L sodium chloride-treated patient skin J Am Acad Dermatol 1991 24 946 951 1869682 Rock I Theoretical considerations In The nature of percuptual adaptation 1966 New York: Basic Books Inc Publishers 250 269 Inoshita T Youngberg GA Artifactual hydropic degeneration in skin biopsy specimens immersed in saline: a light and electron microscopic study Am J Clin Pathol 1983 80 206 209 6349324 Vaughan Jones SA Salas J McGrath JA Palmer I Bhogal BS Black MM A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel's medium Dermatology 1994 189 131 132 8049555 Medenica-Mojsilovic L Fewnske NA Espinoza CG Epidermolysis bullosa acquisita Am J Derm Path 1987 9 324 333

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